CN114867479A

CN114867479A - Methods for treating leukemia and use of leukemia stem cell characteristics prediction for clinical sensitivity to therapy

Info

Publication number: CN114867479A
Application number: CN202080090410.3A
Authority: CN
Inventors: M·波德纳德; K·J·麦克白; D·W·皮尔斯; R·G·卢斯; 樊津红
Original assignee: Celgene Corp
Current assignee: Celgene Corp
Priority date: 2019-10-28
Filing date: 2020-10-27
Publication date: 2022-08-05
Also published as: IL292495A; EP4051277A1; BR112022007932A2; WO2021086829A1; KR20220106976A; CA3155802A1; US20220378773A1; AU2020375794A1; JP2022553427A; MX2022004984A; EP4051277A4

Abstract

Provided herein are methods of using certain biomarkers, such as gene sets (e.g., Leukemia Stem Cell (LSC) characteristics), to predict and monitor clinical sensitivity and therapeutic response to certain compounds in patients with various diseases and disorders, such as cancer (e.g., lymphoma, Multiple Myeloma (MM), and leukemias, such as Acute Myeloid Leukemia (AML)). Also provided herein are methods of treating diseases using the therapeutic compounds.

Description

Methods for treating leukemia and use of leukemia stem cell characteristics prediction for clinical sensitivity to therapy

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional patent application No. 62/927,052 filed on 28.10.2019, which is incorporated herein by reference in its entirety.

1. Field of the invention

In some embodiments, provided herein are methods of using certain biomarkers, such as gene sets (e.g., Leukemia Stem Cell (LSC) characteristics), to predict and monitor clinical sensitivity and therapeutic response to certain compounds in patients with various diseases and disorders, such as cancer (e.g., lymphoma, Multiple Myeloma (MM), and leukemias, such as Acute Myeloid Leukemia (AML)). In certain embodiments, provided herein are methods of treating diseases using the therapeutic compounds.

2. Background of the invention

Cancer is characterized primarily by an increased number of abnormal cells originating from a given normal tissue, invasion of adjacent tissues by these abnormal cells, or lymphatic or blood-borne spread of malignant cells to regional lymph nodes and distant sites (metastasis). Generally, cancers are classified into solid cancers and hematologic cancers. Examples of solid cancers include, but are not limited to, melanoma, adrenal gland cancer, breast cancer, renal cell carcinoma, pancreatic cancer, and Small Cell Lung Cancer (SCLC), among others.

Blood cancers generally include three main types: lymphomas, leukemias, and myelomas. Lymphoma refers to a cancer originating in the lymphatic system. Lymphomas include, but are not limited to, hodgkin's lymphoma, non-hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), and peripheral T-cell lymphoma (PTCL), among others. Leukemia refers to malignant tumors of blood-forming tissues. Acute leukemia involves primarily undifferentiated cell populations, whereas chronic leukemia involves more mature cell forms. Acute leukemias are classified into Acute Lymphoblastic Leukemia (ALL) and Acute Myeloblastic Leukemia (AML) types. Chronic leukemias are classified as Chronic Lymphocytic Leukemia (CLL) or Chronic Myelogenous Leukemia (CML). Myeloma is a cancer of the plasma cells in the bone marrow. Because myeloma often occurs in many parts of the bone marrow, it is commonly referred to as Multiple Myeloma (MM).

Thus, there is a great need for new methods, treatments, and compositions that can be used to treat patients with cancer, including but not limited to lymphoma (e.g., NHL), MM, leukemia (e.g., AML), and solid cancers. Many studies have been conducted with the aim of providing compounds that can be safely and effectively used for the treatment of cancer. For example, we have recently identified certain compounds (e.g., compound D) that are useful for treating cancer, including but not limited to leukemia (e.g., AML). However, there is a need to develop efficient, sensitive and accurate methods to detect, quantify and characterize the pharmacodynamic activity of these compounds. The present invention meets these and other needs.

3. Summary of the invention

Provided herein are methods of identifying a subject with acute myeloid leukemia who is likely to respond to a treatment comprising a compound, or predicting the responsiveness of a subject with or suspected of having AML to a treatment comprising the compound. Also provided herein are methods of treating a subject having AML with a compound.

In one aspect, provided herein is a method of identifying a subject with Acute Myeloid Leukemia (AML) who is likely to respond to a treatment comprising a compound, or predicting the responsiveness of a subject with or suspected of having AML to a treatment comprising the compound, the method comprising:

i. Providing a sample from the subject;

measuring the gene expression level of one or more genes in the sample;

calculating a Leukemia Stem Cell (LSC) signature score for the sample based on the gene expression level of the one or more genes; and

identifying the subject as likely to be responsive to a treatment comprising the compound if the level of the LSC signature score is above its reference level, and the compound is 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide having the structure (compound D):

or a stereoisomer or mixture of stereoisomers thereof, an isotopologue, a pharmaceutically acceptable salt, a tautomer, a solvate, a hydrate, a co-crystal, a clathrate, or a polymorph thereof.

In another aspect, provided herein is a method of treating a subject having AML with a compound, the method comprising:

(a) identifying a subject having AML responsive to treatment comprising said compound, comprising:

i. providing a sample from the subject;

measuring the gene expression level of one or more genes in the sample;

identifying the subject as likely to be responsive to a treatment comprising the compound if the level of the LSC feature score is above its reference level, an

(b) Administering a therapeutically effective amount of the compound to the subject if the subject is identified as likely to be responsive to a treatment comprising the compound, and the compound is compound D or a stereoisomer or a mixture of stereoisomers thereof, an isotopologue, a pharmaceutically acceptable salt, a tautomer, a solvate, a hydrate, a co-crystal, a clathrate, or a polymorph thereof.

In certain embodiments, the LSC feature score is calculated as a weighted sum of the expression levels of the one or more genes.

In certain embodiments, the reference level is the median LSC feature score in the population.

In certain embodiments, the reference level is a predetermined LSC feature score level.

In certain embodiments, a score of a characteristic of LSC above its reference level indicates that the subject has resistant and/or refractory AML.

In certain embodiments, the one or more genes are selected from

(a) CD34, SPINK2, LAPTM48, HOXA5, GUCY1A3, SHANK3, ANGPT1, ARHGAP22, LOC284422, MYCN, MAMDC2, PRSSL1, KIAA0125, GPSM1, HOXA9, MMRN1, FSCN1, DNMT38, HOXA6, AIF1L, SOCS2, CDK6, FAM69B, NGGCP 1, C3orf 387 54, CPXM1, TNFRSF4, ZBTB46, DPYSL3, NYRIN, COL 1, FAM30A, C10orf140, SPNS A, GPRS A, AK 685R A, AKR1C A, FLT A, TFPI A, AHAHS A, 685150, HASHCG A, 685A, 685A, A, 685 DGS A, 685A, 685A, 685 DGS 685 DGS A, 685A, 685A, 685A, 685A, 685A, 685 DGS 685 DGS 685 DGF A, 685A, 685 DGF 685A, 685A, 685A, 685 2K A, 685 2, 685A, 685 2K A, 685 2, 685A, 685A, 685 2, 685A, 685 2, 685S 685A, 685 2, 685A, 685 2, 685A, 685 2, 685 2K 685 2, 685 2K 685A, 685 2, 685A, 685 2, 685D 685 2, 685 2K 685 2, 685 2, 685 2, 685D 685 2, A, 685 2, 685

(b) CD34, SPINK2, LAPTM48, HOXA5, GUCY1A3, SHANK3, ANGPT1, ARHGAP22, LOC284422, MYCN, MAMDC2, PRSSL1, KIAA0125, GPSM1, HOXA9, MMRN1, FSCN1, DNMT38, HOXA6, AIF1L, SOCS2, CDK6, FAM69B, NGFRAP1, C3orf54, CPXM1, TNFRSF4, ZBTB4, DPYSL 4, NYRRIN, COL24A 4, FAM30 4, C10orf140, SPNS 4, GPR 4, AKR1C 4, FLT 4, TFPI 4, KCR 4, C150, VWF 4, ATP 4,

ATP

4, 4 and AFVM 4.

In certain embodiments, the one or more genes are selected from AKR1C3, ARHGAP22, CD34, CDK6, CPXM1, DNMT3B, DPYSL3, EMP1, GPR56, KIAA0125, lamtm 4B, MMRN1, NGFRAP1, NYNRIN, SMIM24, SOCS2, and ZBTB 46.

In certain embodiments, the LSC trait score is based on the gene expression level of AKR1C3, ARHGAP22, CD34, CDK6, CPXM1, DNMT3B, DPYSL3, EMP1, GPR56, KIAA0125, lamtm 4B, MMRN1, NGFRAP1, NYNRIN, SMIM24, SOCS2, and ZBTB 46.

In certain embodiments, the LSC feature score is calculated as follows: (the expression level of DNMT3B × the weight of DNMTT 3B) + (the expression level of ZBTB46 × the weight of ZBTB 46) + (the expression level of NYNRIN × the weight of NYNRIN) + (the expression level of ARHGAP22 × the weight of ARHGAP 22) + (the expression level of lamm 4B × the weight of lamm 4B) + (the expression level of MMRN1 × the weight of MMRN 1) + (the expression level of DPYSL 1 × the weight of DPYSL 1) + (the expression level of KIAA0125 × the weight of KIAA 0125) + (the expression level of CDK 1 × the weight of CDK 1) + (the expression level of CPXM1 × the weight of CPXM 1) + (the expression level of SOCS 1 × the weight of SOCS 1) + (the expression level of SMIM 1 × the weight of SMIM 1) + (the weight of emgpr 1 × the expression level of emgpr 1 × the weight of SOCS 1) + (the expression level of samakc 1 × the expression level of samm 1) + (the weight of samr 1) + (the expression level of samgpr 1 × the weight of samm 1) + (the weight of ZBTB 1) + (the expression level of ZBTB 1 × the weight of ZBTB 1) + (the expression level of ZBTB 1) + (the weight of ZBTB 1) + (the expression level of ZBTB 1) + (the weight of ZBTB 1); and DNMTT3B is within the range of 0.08 to 0.09, ZBTB46 is within the range of-0.03 to-0.04, NYNRIN is within the range of-0.008 to 0.009, ARHGAP22 is within the range of-0.015 to 0.01, lamtm 4B is within the range of-0.006 to 0.005, MMRN1 is within the range of 0.02 to 0.03, DPYSL3 is within the range of 0.02 to 0.03, KIAA0125 is within the range of 0.01 to 0.02, CDK6 is within the range of-0.08 to-0.07, CPXM1 is within the range of-0.02 to-0.03, SOCS2 is within the range of 0.02 to 0.03, SOCS 3 is within the range of 0.02 to 0.03, emm 3 is within the range of 0.04, emm 0.05 to 0.05, gprn 3 is within the range of-0.04, and GPR 0.04 to 0.05 is within the range of-0.04, and emr 3 is within the range of-0.04, and emr 0.04.

In certain embodiments, the LSC feature score is calculated as follows: (expression level of DNMT3B × 0.0874) + (expression level of ZBTB46 × -0.0347) + (NYNRIN × 0.00865) + (ARHGAP22 × -0.0138) + (lamm 4B expression level × 0.00582) + (MMRN1 × 0.0258) + (DPYSL3 expression level × 0.0284) + (KIAA0125 expression level × 0.0196) + (CDK6 expression level of × -0.0704) + (CPXM1 expression level × -0.0258) + (SOCS2 × 0.0271) + (SMIM24 expression level × -0.0226) + (EMP1 expression level × 0.0146) + (NGFRAP1 × 0.0465) + (CD34 expression level × 0.0338) + (AKR1 × 3) + (akgpr × 3646) + (akgpr 3646).

In certain embodiments, the reference level is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8.0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.

In certain embodiments, the LSC signature score is based on the gene expression levels of TNFRSF4, SLC4a1, SLC7a7 and AIM 2.

In certain embodiments, the LSC feature score is calculated as follows: (expression level of TNFRSF4 × weight of TNFRSF 4) + (expression level of SLC4a1 × weight of SLC4a 1) + (expression level of SLC7a7 × weight of SLC7a 7) + (expression level of AIM2 × weight of AIM 2); and TNFRSF4 is weighted in the range of-1.5 to-1, SLC4A1 is weighted in the range of 13 to 14, SLC7A7 is weighted in the range of-4 to-3, and AIM2 is weighted in the range of-3 to-4.

In certain embodiments, the LSC feature score is calculated as follows: (expression level of TNFRSF4 × -1.13) + (expression level of SLC4A1 × 13.59) + (expression level of SLC7A7 × -3.57) + (expression level of AIM2 × -3.04).

In certain embodiments, the reference level is in the range of-50 to 115, -45 to 110, -40 to 105, -37 to 100, -30 to 95, -25 to 90, -20 to 85, -15 to 80, -10 to 75, -5 to 70, 0 to 65, 5 to 60, 10 to 55, 15 to 50, 20 to 45, 25 to 40, or 30 to 35.

In certain embodiments, the LSC signature score is based on the gene expression levels of SLC4a1, SLC7a7, and AIM 2.

In certain embodiments, the LSC feature score is calculated as follows: (SLC4a1 expression level x weight of SLC4a 1) + (SLC7a7 expression level x weight of SLC7a 7) + (AIM2 expression level x AIM2 weight); and SLC4A1 is weighted in the range of 11 to 15, SLC7A7 is weighted in the range of-5.5 to-1.5, and AIM2 is weighted in the range of-5 to-1.

In certain embodiments, the LSC feature score is calculated as follows:

the following calculations were made: (SLC4A1 expression level X13.59) + (SLC7A7 expression level X-3.57) + (AIM2 expression level X-3.04).

In certain embodiments, the reference level is in the range of-65 to 110, -60 to 105, -55 to 100, -49 to 93, -45 to 90, -40 to 85, -35 to 80, -30 to 75, -25 to 70, -20 to 65, -15 to 60, -10 to 55, -5 to 50, 0 to 45, 5 to 40, 10 to 35, 15 to 30, 20 to 35, or 25 to 30.

In another aspect, provided herein is a method of identifying a subject having AML who is likely to respond to a treatment comprising a compound, or predicting the responsiveness of a subject having or suspected of having AML to a treatment comprising the compound, the method comprising:

i. providing a sample from the subject;

administering the compound to the sample;

measuring the proportion of one or more cell types;

identifying the subject as likely to be responsive to a treatment comprising the compound if the ratio of the one or more cell types is different from the reference ratio of the cells, and the compound is compound D or a stereoisomer or a mixture of stereoisomers thereof, an isotopologue, a pharmaceutically acceptable salt, a tautomer, a solvate, a hydrate, a co-crystal, a clathrate, or a polymorph thereof.

i. providing a sample from the subject;

administering the compound to the sample;

Measuring the proportion of one or more cell types;

identifying the subject as likely to be responsive to a treatment comprising the compound if the proportion of the one or more cell types is different from the reference proportion of the cells, and

In certain embodiments, the reference proportion of a cell type is the proportion of said cell type in said sample prior to administration of said compound.

In certain embodiments, the reference ratio for a cell type is a predetermined ratio.

In certain embodiments, the method comprises measuring the proportion of primitive cells and/or the proportion of differentiated leukemia cells.

In certain embodiments, a decrease in the proportion of blasts and/or an increase in the proportion of differentiated leukemia cells as compared to their respective proportions prior to administration of the compound indicates that the subject is likely to be responsive to a treatment comprising the compound.

In certain embodiments, the method comprises measuring the proportion of CD34+, CD15+, CD14+ and/or CD11b + cells.

In certain embodiments, the method comprises measuring the proportion of CD34+ cells, and a decrease in the proportion of CD34+ cells as compared to the proportion of CD34+ cells prior to administration of the compound indicates that the subject is likely to be responsive to a treatment comprising the compound.

In certain embodiments, the method comprises measuring the proportion of CD15+ cells and/or CD14+ cells, and an increase in the proportion of CD15+ cells and/or CD14+ cells as compared to the proportion of CD15+ cells and/or CD14+ cells prior to administration of the compound indicates that the subject is likely to be responsive to a treatment comprising the compound.

In certain embodiments, the AML is refractory or resistant.

In certain embodiments, the AML is resistant to treatment with one or more agents selected from daunomycin, cytarabine (ara-C) and gemtuzumab ozogamicin or resistant to chemotherapy.

4. Description of the drawings

Figures 1A-1C depict compound D mediated degradation of GSPT1 in acute myeloid cells in vitro. Figure 1A shows the degradation of gstt 1 as assessed by flow cytometry analysis using anti-gstt-1 conjugated antibodies that bind to gstt 1, measured by the MFI of Alexa flur 647 fluorophore, after 4 hours of in vitro incubation of compound D with different LSC 17-scored indicative AML patient samples. Figure 1B shows the degradation of gstt 1 as assessed by flow cytometry analysis using anti-gstt-1 conjugated antibodies that bind to GSPT1, measured by the MFI of Alexa flour647 fluorophore, after 24 hours in vitro incubation of compound D with different LSC17 scored indicative AML patient samples. Results are expressed as a percentage of vehicle control (1.0 corresponds to 100%). Figure 1C shows the degradation of GSPT1 after 24 hours of incubation with 100nM compound D, as evaluated for AML patient samples receiving high and low LSC17 scores, with the results presented as mean values with error bars representing the standard error of the mean. GSPT1 ═ G1 to S phase change protein 1; ID is identification; LSC17 ═ leukemia stem cell 17-gene characteristics; MFI-median fluorescence intensity; nd is undetermined.

Figures 2A-2C depict compound D-mediated induction of apoptosis in primary acute myeloid leukemia blasts in vitro. Figure 2A shows a representative flow cytometry dot plot for determination of apoptotic cells (upper panel shows apoptosis as assessed by FSC/SSC gated cells; lower panel shows apoptosis as assessed by annexin V staining after vehicle (0nM) or 100nM compound D treatment). Figure 2B shows the percentage of annexin V + cells (apoptotic) assessed after incubation of 9 AML patient samples with compound D (0, 3, 30 or 100 nM). Samples were grouped by high and low LSC17 score. Figure 2C shows the total cell number evaluated for 9 AML patient samples after incubation with compound D (0, 3, 30 or 100 nM). Samples were grouped by high and low LSC17 scores. Data are shown as group means with error bars indicating the standard error of the means. The P value represents a statistical comparison between the LSC17 high group and the LSC17 low group. FSC — forward scattering; LSC17 ═ leukemia stem cell 17-gene characteristics; SSC ═ side scatter; 7AAD ═ 7-amino actinomycin D.

Figure 3 depicts compound D mediated inhibition of colony formation of leukemic progenitors. Colony numbers formed at 24 hours were evaluated per 100,000 cells for 9 AML patient samples after incubation with compound D (0, 3, 30 or 100nM), where colony reduction in the colony forming samples was evaluated as a percentage of vehicle control when samples were grouped by high and low LSC17 scores. The results are shown as group means, with error bars indicating the standard error of the means. ID is identification; LSC17 ═ leukemia stem cell 17-gene characteristics.

Figure 4 depicts the effect of compound D on acute myeloid leukemia patient 110500 and patient 90191 xenografts. The percentage of AML cells from the Right Femur (RF) or from the left femur + tibial Bone Marrow (BM) or AML cells with different markers (CD34+ or CD15+) after treatment with different doses of compound D is plotted. P values represent statistical comparisons with vehicle controls of the same bone marrow origin. AML ═ acute myeloid leukemia; bid is twice a day; BM ═ bone marrow (not injected); qd-daily; RF-right femur (AML cell injection).

Figure 5 depicts the reactivity to compound D and the variation of different cell types in samples with high LSC17 signature scores. The percentage of AML cells or the percentage of CD34+ cells in RF or BM and the absolute number of AML or CD34+ cells from 3 AML samples with a high LSC17 score are shown. The circle symbols represent data from vehicle control mice, and the square symbols represent compound D treated mice. P values represent statistical comparisons between compound D treatment and vehicle controls. AML ═ acute myeloid leukemia; BM ═ bone marrow (not injected); LSC17 ═ leukemia stem cell 17-gene characteristics; RF-right femur (AML cell injection).

Figure 6 depicts the reactivity to compound D and the variation of different cell types in samples with low LSC17 signature scores. The percentage of AML cells or the percentage of CD34+ cells in RF or BM and the absolute number of AML or CD34+ cells from three AML samples with low LSC17 scores are shown. The circle symbols represent data from vehicle control mice, and the square symbols represent compound D treated mice. P values represent statistical comparisons between compound D treatment and vehicle controls. AML ═ acute myeloid leukemia; BM ═ bone marrow (not injected); LSC17 ═ leukemia stem cell 17-gene characteristics; RF-right femur (AML cell injection).

Figures 7A-7E depict secondary transplants following compound D treatment of primary transplanted mice. Fig. 7A shows the percentage of engrafted AML cells from bone marrow (RF or BM) from secondary mice injected with cells of AML patient sample 110590 isolated from bone marrow of primary mice dosed with 2.5mg/kg compound D. Limiting Dilution Assay (LDA) analysis (lower panel) showed a 13.3-fold reduction in Leukemic Stem Cells (LSCs) after compound D administration compared to vehicle (lower panel). Figure 7B shows the percentage of engrafted AML cells from bone marrow (RF or BM) from secondary mice injected with cells from AML patient sample 120860 isolated from the bone marrow of primary mice dosed with 2.5mg/kg compound D. Limiting Dilution Assay (LDA) analysis (lower panel) showed no difference in LSC frequency. Fig. 7C shows the percentage of engrafted AML cells from bone marrow (RF) from secondary mice injected with cells of AML patient sample 100348 isolated from the bone marrow of primary mice dosed with 2.5mg/kg compound D. Figure 7D shows the percentage of CD45+ cells in isolated bone marrow from once-treated mice for each patient sample. Total cells and total AML cells injected into each secondary mouse (without mouse cell depletion) are also shown. Fig. 7E shows the percentage of AML grafts in secondary mice that received cells from AML patient sample 110102 (top) or AML patient sample 0590 (bottom) from vehicle or compound D treated primary xenograft mice, showing that each symbol represents a single secondary transplant mouse. AML ═ acute myeloid leukemia; BM ═ bone marrow (not injected); ID is identification; k is thousand; m is million; RF-right femur (AML cell injection).

FIGS. 8A-8B depict a representative flow cytometry analysis of cord blood xenografts. Figure 8A shows a representative gating strategy for identifying cell populations isolated from cord blood xenograft vehicles. Figure 8B shows a representative gating strategy for identifying cell populations isolated from compound D-treated mouse bone marrow. CD45+ cells were gated (GlyA-CD45+, left column) and further sub-gated to determine CD38 and CD34 expression (second left column) or CD19 and CD33 expression (middle column). GlyA-CD45+ and GlyA-CD45+ CD33+ cells were sub-gated to determine CD14 and CD15 expression (second and rightmost columns from right, respectively). BM ═ bone marrow (not injected); GlyA ═ glycophorin a; RF ═ right femur (acute myeloid leukemia cell injection).

Figures 9A-9D depict the effect of compound D on umbilical cord blood graft populations. The percentage or absolute cell number of each subcellular type was plotted for CB1 and CB2 engrafted cells. Fig. 9A shows the percentage or absolute cell number of CB1 and CB2 engrafted cells. Figure 9B shows the percentage or absolute number of CD19+ or CD33+ cells. Figure 9C shows the percentage or absolute number of CD15+ or CD14+ cells in CD45+ grafts. Figure 9D shows the percentage or absolute number of GlyA + cells. The circle symbols represent data from vehicle control treated mice, and the square symbols represent data from compound D treated mice. P values represent statistical comparison of compound D treatment to control. BM ═ bone marrow (not injected); CB ═ cord blood; GlyA ═ glycophorin a; RF-right femur (AML cell injection).

FIGS. 10A-10D depict the effect of Compound D on CD34+ and CD34+/CD 38-blasts. For RF or BM, the percentage or absolute number of each subcellular type was plotted for CB1 and CB2 implanted cells. Figure 10A shows the percentage or absolute number of CD34+ cells. FIG. 10B shows the percentage or absolute number of CD34+/CD 38-cells. FIG. 10C shows the percentage or absolute number of CD34+/CD19+ cells. FIG. 10D shows the percentage or absolute number of CD34+/CD33+ cells. The circle symbols represent data from vehicle control treated mice, and the square symbols represent data from compound D treated mice. P values represent statistical comparison of compound D treatment to control. BM ═ bone marrow (not injected); CB ═ cord blood; RF-right femur (AML cell injection).

FIG. 11 depicts the effect of Compound D on acute myeloid leukemia grafts in NOD/SCID mice. Human CD45+/CD33+ AML implantation in injected femur (RF, top panel) and non-injected bone (BM, bottom panel) of compound D (squares) or vehicle control (circles) treated mice is summarized. Each symbol indicates the level of engraftment in each treated mouse, and the bars indicate the median value for each treatment group. AML ═ acute myeloid leukemia; BM ═ bone marrow (no bone injected); ns is not significant; RF ═ right femur (injected bone). P < 0.05; p < 0.01; p < 0.001; p < 0.0001.

Figures 12A-12E depict phenotype profiles induced by compound D administration. Figure 12A shows representative flow cytometric analysis of cell surface markers CD15, CD14, CD34, and CD38 on leukemia cells in AML transplants after compound D or vehicle treatment. Figure 12B shows representative flow cytometric analysis of cell surface markers CD15, CD14, CD34, CD11B, and CD38 on leukemia cells in AML transplants after compound D or vehicle treatment. Figure 12C shows the percentage of CD34+ cells from AML grafts after compound D (squares) or vehicle control (circles) treatment. Figure 12D shows the percentage of CD15+ cells from AML grafts after compound D (squares) or vehicle control (circles) treatment. Figure 12E shows the percentage of CD14+ cells from AML grafts after compound D (squares) or vehicle control (circles) treatment. Samples were grouped into 3 classes: increase (upper panel), decrease (middle panel) and no change (lower panel) in CD34+, CD15+, and CD14+ cells. Each symbol indicates the percentage of the corresponding population in AML graft per treated mouse. The percentage in parentheses after each patient number is the relative reduction caused by compound D treatment to indicate the responsiveness of each sample to the drug. Bars indicate mean values. AML ═ acute myeloid leukemia; BM ═ bone marrow (no bone injected); ns is not significant; RF ═ right femur (injected bone). P < 0.05; p < 0.01; p < 0.001; p < 0.0001.

Figure 13 depicts the heterogeneous response to compound D and correlation with LSC17 scores in primary acute myeloid leukemia grafts. The effect of compound D on Acute Myeloid Leukemia (AML) transplants was presented as a percentage reduction in AML versus vehicle control treatment. Each symbol represents the relative reduction in median AML implantation for each patient sample, with the long horizontal bars indicating the mean of each group and the shorter horizontal bars indicating the Standard Error (SEM) of the mean. The samples were summed together (filled circles) and grouped into high LSC17 (squares) and low LSC17 scores (triangles). BM ═ bone marrow (no bone injected); LSC ═ leukemic stem cells; RF ═ right femur (injected bone).

Figures 14A-14C depict LSC4 gene signature and LSC3 gene signature and predictions of their responsiveness to compound D treatment. Gene expression profiles were generated from primary cells of each patient sample by RNA-Seq. Fig. 14A shows the 4-gene scores (LSC4) identified in the 89 LSC-associated gene set. The solid curve represents the reduction obtained by the drug in the experiment, and the dashed curve represents the% reduction predicted by the 4-gene score. Fig. 14B shows the discretization of the prediction by median threshold as "reaction" or "no reaction" and the correlation between score and% reduction (r 0.87, p 0.02). The solid curve represents the reduction obtained by the drug in the experiment, and the dashed curve represents the% reduction prediction obtained by the 4-gene score. FIG. 14C shows the signal at about 46 LSCs ^- The 3-gene score (LSC3) identified in the gene set, which can predict the reactivity to compound D, and the results are very similar to the LSC4 gene signature described above. The solid curve represents the reduction obtained by the drug in the experiment, and the dashed curve represents the% reduction prediction obtained by the 3-gene score.

Figures 15A-15D depict clinical features of patients and graft responsiveness to compound D. Figure 15A depicts a profile based on its new and secondary/relapses, characterizing the sample for its response to compound D. Figure 15B depicts a profile based on its poor and intermediate prognosis, characterizing the sample for its response to compound D. Figure 15C depicts a profile based on its cytogenetically normal and abnormal karyotypes, characterizing the sample for its response to compound D. FIG. 15D depicts a sample characterized for its response to Compound D based on the profile of its Flt3-ITD versus wild type Flt3 in cytogenetically normal AML. Each symbol represents the relative reduction in median AML implantation for each patient sample, and the bars indicate median values. AML ═ acute myeloid leukemia; BM ═ bone marrow (no bone injected); CN-AML ═ cytogenetically normal acute myeloid leukemia; flt3-ITD ═ fms-like tyrosine kinase 3-internal tandem repeats; RF ═ right femur (injected bone).

Figures 16A-16D depict that compound D induces apoptosis in acute myeloid leukemia cells in vitro by reducing GSPT 1. Acute myeloid leukemia cells were cultured in vitro in medium supplemented with growth factors at different concentrations of compound D. Figure 16A shows the degradation of GSPT1 in primary leukemia cells upon 24 hours exposure to compound D. Fig. 16B shows induction of apoptosis in leukemia cells. Figure 16C shows the reduction of viable cells after treatment with compound D. Figure 16D shows the depletion of colony forming leukemic progenitors by compound D. GSPT1 ═ G1 to S phase change protein 1.

Figure 17 depicts the induction of apoptosis and cell death by compound D treatment. Apoptosis and cell death in mice treated with compound D were assessed by staining cells with propidium iodide. Each symbol indicates the percentage of PI + events in individual mice treated with vehicle (circles) or compound D (squares). The bars indicate the median value. BM ═ bone marrow (no bone injected); PI ═ propidium iodide; RF ═ right femur (injected bone). P < 0.05; p < 0.01; p < 0.001; p < 0.0001.

Figures 18A-18B depict treatment with compound D to degrade GSPT1 in acute myeloid leukemia grafts. Intracellular flow cytometry (FACS) was performed to measure expression of GSPT1 following 3-dose compound D treatment of AML-bearing mice. Figure 18A shows the mean fluorescence intensity of GSPT1 in CD33+ AML cells harvested from injected RF (top panel) and non-injected BM (bottom panel) mice treated with vehicle (circles) and compound D (squares). Each symbol indicates data from individual mice treated with vehicle (circles) or compound D (squares), and bars indicate median values. The numbers above the data points are the p-values between compound D treatment and control. Figure 18B shows the relative reduction in GSPT1 by compound D. Each bar indicates the percentage of median GSPT1 MFI of compound D treatment relative to vehicle control. The percentage in parentheses after each patient number is the relative reduction caused by 4 weeks of compound D treatment to indicate the responsiveness of each sample to the drug. AML ═ acute myeloid leukemia; BM ═ bone marrow (no bone injected); GSPT1 ═ G1 to S phase change protein 1; MFI ═ mean fluorescence intensity; PI ═ propidium iodide; RF ═ right femur (injected bone).

Figure 19 depicts a representative secondary transplant limiting dilution assay (confidence interval plot of leukemic stem cell frequency). The solid line indicates the average estimate of LSC frequency, and the dashed lines indicate the lower and upper limits of the estimation of LSC frequency in vehicle control (grey dashed line) or compound D (black dashed line) one mouse. Each individual symbol indicates the log of the unresponsive fraction associated with each cell dose. LSC ═ leukemic stem cells; veh is vehicle.

5. Detailed description of the preferred embodiments

Certain compounds provided herein (including compound D) are cereblon E3 ligase modulators. For example, compound D caused degradation of translation termination factor G1 to S phase change protein 1(GSPT1) and resulted in a combined stress response, Unfolded Protein Response (UPR) activation, and apoptosis in Acute Myeloid Leukemia (AML) cells. Compound D is in clinical development of relapsed and refractory AML.

The refractory nature to induction chemotherapy and relapse after achieving remission are major obstacles to cure AML. After standard induction chemotherapy, patients are assigned to different post-remission strategies based on cytogenetic and molecular abnormalities that broadly define adverse, intermediate, and favorable risk categories. However, some patients do not respond to induction therapy, and yet another subset will eventually relapse despite the lack of adverse risk factors. There is an urgent need for better biomarkers to identify these high risk patients and treatments for this group of patients.

To develop predictive and/or prognostic biomarkers associated with sternness, Ng et al (Ng SW et al Nature.2016; 540(7633):433-37) generated a 17-gene score (LSC17 score) using a functional leukemia stem cell population. More details regarding the method of generating the LSC17 score are described in section 6.1. As shown by Ng et al, patients with a high LSC17 score had poor outcome with current treatments, including allogeneic stem cell transplantation.

Surprisingly, in the present study described in section 6.2, AML samples with a high LSC17 score were more sensitive to compound D treatment than samples with a low LSC17 score. Furthermore, in the study described in section 6.3, most samples with high LSC17 scores responded well to compound D, and more than half of it was eradicated in mouse bone marrow.

The unexpected observations provided herein indicate that compound D can be used to treat AML patients with more aggressive disease in the context of primary induction therapy and/or refractory AML patients that are resistant to conventional treatments such as chemotherapy.

In addition, as shown in section 6, the present disclosure also identifies cell surface markers or changes thereof that can be used to predict responsiveness to a therapeutic compound (e.g., compound D, or a stereoisomer or a mixture of stereoisomers thereof, a tautomer, a pharmaceutically acceptable salt, a solvate, an isotopologue, a prodrug, a hydrate, a co-crystal, a clathrate, or a polymorph thereof).

5.1. Definition of

As used herein, the term "cancer" includes, but is not limited to, solid cancers and cancers of the hematologic system. The term "cancer" refers to a disease of a tissue or organ, including, but not limited to, bladder cancer, bone cancer, blood cancer, brain cancer, breast cancer, cervical cancer, chest cancer, colon cancer, endometrial cancer, esophageal cancer, eye cancer, head cancer, kidney cancer, liver cancer, lymph node cancer, lung cancer, oral cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, larynx cancer, and uterine cancer. Specific cancers include, but are not limited to, advanced malignant tumors, amyloidosis, neuroblastoma, meningioma, hemangiopericyte tumor, multiple brain metastases, glioblastoma multiforme, glioblastoma, brain stem glioma, pre-refractory malignant brain tumor, glioblastoma, recurrent malignant glioma, anaplastic astrocytoma, anaplastic oligodendroglioma, neuroendocrine tumor, rectal adenocarcinoma, unresectable colorectal cancer, metastatic hepatocellular carcinoma, kaposi's sarcoma, nuclear acute myeloblastic leukemia, hodgkin's lymphoma, non-hodgkin's lymphoma, cutaneous T-cell lymphoma, cutaneous B-cell lymphoma, diffuse large B-cell lymphoma, low-grade follicular lymphoma, malignant melanoma, malignant mesothelioma, malignant pleural effusion mesothelioma syndrome, peritoneal carcinoma, papillary serous carcinoma, peritoneal carcinoma, multiple brain metastases, colorectal carcinoma, neuroblastoma, colorectal carcinoma, or carcinoma or a patient Gynecological sarcomas, soft tissue sarcomas, scleroderma, cutaneous vasculitis, Langerhans' cell histiocytosis, leiomyosarcoma, progressive osteogenic fibrodysplasia, hormone refractory prostate cancer, resected high risk soft tissue sarcomas, unresectable hepatocellular carcinoma, Fahrenheit macroglobulinemia, smoldering myeloma, indolent myeloma, fallopian tube carcinoma, androgen-independent prostate cancer, androgen-dependent stage IV non-metastatic prostate cancer, hormone-insensitive prostate cancer, thyroid papillary carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, and leiomyoma.

As used herein, "hematological cancer" includes myeloma, lymphoma and leukemia. In one embodiment, the myeloma is multiple myeloma. In some embodiments, the leukemia is, for example, Acute Myelogenous Leukemia (AML), Acute Lymphocytic Leukemia (ALL), adult T-cell leukemia, Chronic Lymphocytic Leukemia (CLL), hairy cell leukemia, myelodysplasia, myeloproliferative disorders, Chronic Myelogenous Leukemia (CML), myelodysplastic syndrome (MDS), human T-lymphotropic virus type 1 (HTLV-1) leukemia, mastocytosis, or B-cell acute lymphoblastic leukemia. In some embodiments, the lymphoma is, for example, diffuse large B-cell lymphoma (DLBCL), B-cell immunoblastic lymphoma, small non-dividing cell lymphoma, human T-lymphotropic virus type 1 (HTLV-1) leukemia/lymphoma, adult T-cell lymphoma, peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma (CTCL), Mantle Cell Lymphoma (MCL), Hodgkin Lymphoma (HL), non-Hodgkin lymphoma (NHL), AIDS-related lymphoma, follicular lymphoma, small lymphocytic lymphoma, large B-cell lymphoma enriched for T cells/histiocytes, transformed lymphoma, primary mediastinal (thymic) large B-cell lymphoma, splenic marginal zone lymphoma, Rickett's transformation, nodular marginal zone lymphoma, or ALK-positive large B-cell lymphoma. In one embodiment, the hematologic cancer is indolent lymphoma, including, for example, DLBCL, follicular lymphoma, or marginal zone lymphoma.

When used in conjunction with cancer, the term "prognostic risk" refers to the possible outcome of a cancer, including responsiveness to certain treatments, duration or degree of remission, potential survival rate, likelihood of relapse, and the like. Factors that affect a patient's prognostic risk include, but are not limited to, demographics (e.g., age, race, sex, etc.), disease specificity (e.g., stage of cancer), inheritance (e.g., risk genes), co-morbidity (e.g., other conditions that accompany cancer), and the like. A good "prognostic risk" means that the patient is likely to respond to certain treatments, likely to survive, and/or less likely to relapse, etc. By poor "prognostic risk" is meant that the patient is less likely to respond to certain treatments, less likely to survive, and/or likely to relapse, etc.

As used herein and unless otherwise indicated, the terms "treatment", "treating" and "treatment" refer to activities that occur when a patient has a particular cancer, which reduce the severity of the cancer or impede or slow the progression of the cancer.

The term "sensitive" or "sensitive" when referring to treatment with a compound is a relative term that refers to the degree of effectiveness of the compound in reducing or diminishing the progression of a tumor or disease being treated. For example, when used in reference to a cell or tumor treatment with which a compound is associated, the term "increased sensitivity" refers to an increase in the effectiveness of the tumor treatment of at least about 5% or more.

As used herein, the terms "compound" and "therapeutic compound" are used interchangeably and include the non-limiting examples of compounds disclosed in section 5.5 below.

As used herein and unless otherwise specified, the term "therapeutically effective amount" of a compound is an amount sufficient to provide a therapeutic benefit in treating or managing cancer or to delay or minimize one or more symptoms associated with the presence of cancer. A therapeutically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in treating or managing cancer. The term "therapeutically effective amount" can encompass an amount that improves the overall therapy of the cancer, alleviates or avoids a symptom or cause of the cancer, or enhances the therapeutic efficacy of another therapeutic agent. The term also refers to an amount of a compound that is sufficient to elicit the biological or medical response that is being sought by a researcher, veterinarian, medical doctor or clinician of a biomolecule (e.g., protein, enzyme, RNA or DNA), cell, tissue, system, animal or human.

The term "responsiveness" or "response" when used in relation to treatment refers to the degree of effectiveness of the treatment in alleviating or reducing the symptoms of the disease being treated (e.g., cancer, such as MM or AML). For example, the term "increased responsiveness" when used in relation to treatment of a cell or subject refers to an increase in effectiveness in alleviating or reducing a symptom of a disease as compared to a reference treatment (e.g., of the same cell or subject or of a different cell or subject) when measured using any method known in the art. In certain embodiments, the increase in effectiveness is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%.

An improvement in cancer or cancer-related disease can be characterized as a complete or partial response. By "complete response" is meant the absence of a clinically detectable disease, in which any prior abnormal radiographic studies, bone marrow and cerebrospinal fluid (CSF) or abnormal monoclonal protein measurements were normalized. By "partial response" is meant that all measurable tumor burden (i.e., the number of malignant cells present in the subject, or the measured tumor mass volume or amount of abnormal monoclonal protein) is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% in the absence of new lesions. The term "treatment" contemplates both complete and partial responses.

The term "likelihood" generally refers to an increase in the probability of an event. The term "likelihood" when used in relation to the effectiveness of a patient's tumor response generally contemplates an increased likelihood that tumor progression or tumor cell growth rate will be reduced. The term "likelihood" when used in relation to the effectiveness of a patient's tumor response may also generally mean an increase in an indicator, such as mRNA or protein expression, which may evidence an increase in the progress of tumor therapy.

The term "predictive" generally means predetermined or identified. For example, when used to "predict" the effectiveness of a cancer treatment, the term "predict" can mean that the likelihood of the outcome of the cancer treatment can be determined at the beginning, before the beginning of the treatment, or before significant progression of the treatment period.

As used herein, the term "monitoring" generally refers to monitoring, supervision, observation, tracking, or inspection of an activity. For example, the term "monitoring the effectiveness of a compound" refers to tracking the effectiveness of treating cancer in a patient or tumor cell culture. Similarly, the term "monitoring" as used in reference to patient compliance, either alone or in a clinical trial, refers to tracking or confirming that a patient is actually taking the medication being tested as prescribed. For example, monitoring can be performed by tracking the expression of mRNA or protein biomarkers.

As used herein, the term "modulate" refers to controlling a molecular activity or biological function, such as enhancing or diminishing an activity or function.

The term "refractory" or "resistant" refers to a condition in which a patient has residual cancer cells (e.g., leukemia or lymphoma cells) in their lymphatic system, blood, and/or hematopoietic tissues (e.g., bone marrow) even after intensive therapy.

A "biomarker" or "biomarker" is a substance whose detection indicates a particular biological state, such as for example the presence of cancer. In some embodiments, the biomarkers may be determined individually. In other embodiments, several biomarkers may be measured simultaneously. In some embodiments, a "biomarker" indicates a change in mRNA expression levels that may be correlated with risk or progression of a disease or with susceptibility of a disease to a given treatment. In some embodiments, the biomarker is a nucleic acid, such as mRNA or cDNA. In additional embodiments, a "biomarker" indicates a change in the level of polypeptide or protein expression that may be associated with the risk or progression of a disease or with the sensitivity of a patient to treatment. In some embodiments, the biomarker may be a polypeptide or protein or fragment thereof. The relative levels of a particular protein can be determined by methods known in the art. For example, antibody-based methods such as immunoblotting, enzyme-linked immunosorbent assay (ELISA), or other methods may be used.

As used herein, "gene set" refers to one or more genes selected by one of skill in the art. Genes may be based on their relationship to each other; their association with certain cell types, biological functions, phenotypes, or cellular pathways, etc.; or merely grouped according to the discretion of a person skilled in the art. As used herein, a gene set may comprise as few as just one gene or as many as hundreds, thousands, or hundreds of thousands of genes.

As used herein, "signature" or "gene signature" refers to a group of genes. In some embodiments, the set of genes are related to each other in that they are associated with certain cell types, biological functions, phenotypes, or cellular pathways, and the like. The characteristics may be defined by the skilled person on the basis of different experimental data and/or statistical analysis methods, i.e. a particular characteristic may contain a different number of genes or a different particular gene, depending on the criteria chosen by the skilled person. An example of a genetic signature is the LSC signature.

As used herein, "LSC 17" or "LSC 17 signature" refers to a genetic signature comprising the following 17 genes: AKR1C3, ARHGAP22, CD34, CDK6, CPXM1, DNMT3B, DPYSL3, EMP1, GPR56, KIAA0125, lamm 4B, MMRN1, NGFRAP1, NYNRIN, SMIM24, SOCS2, and ZBTB 46. As used herein, "LSC 4" or "LSC 4 signature" refers to a genetic signature comprising 4 genes: TNFRSF4, SLC4A1, SLC7A7, and AIM 2. As used herein, "LSC 3" or "LSC 3 signature" refers to a genetic signature comprising the following 3 genes: SLC4A1, SLC7A7, and AIM 2. As used herein, "LSC 17 score" or "LSC 17 trait score" refers to a score based on the expression level of LSC17 trait comprising the following 17 genes: AKR1C3, ARHGAP22, CD34, CDK6, CPXM1, DNMT3B, DPYSL3, EMP1, GPR56, KIAA0125, lamm 4B, MMRN1, NGFRAP1, NYNRIN, SMIM24, SOCS2, and ZBTB 46. Similarly, as used herein, a "LSC 4 score" or "LSC 4 trait score" is a score calculated based on the expression level of the LSC4 trait described above. As used herein, a "LSC 3 score" or "LSC 3 trait score" is a score calculated based on the expression level of the LSC3 trait described above.

The terms "polypeptide" and "protein" as used interchangeably herein refer to a polymer of three or more amino acids linked by peptide bonds in a sequence array. The term "polypeptide" includes proteins, protein fragments, protein analogs, oligopeptides and the like. The term "polypeptide" as used herein may also refer to a peptide. The amino acids that make up the polypeptide may be naturally derived or may be synthetic. The polypeptide may be purified from a biological sample. Polypeptides, proteins or peptides also encompass modified polypeptides, proteins and peptides, such as glycopolypeptides, glycoproteins or glycopeptides; or a lipopeptide, lipoprotein, or lipopeptide.

As used herein, the term "expressed" or "expression" refers to the transcription from a gene to produce an RNA nucleic acid molecule that is at least partially complementary to a region of one of the two nucleic acid strands of the gene. As used herein, the term "expressed" or "expression" also refers to translation from an RNA molecule to produce a protein, polypeptide, or portion thereof.

The term "expression level" refers to the number, accumulation, or rate of biomarker molecules or gene sets. The expression level may be expressed, for example, by the amount or rate of synthesis of messenger rna (mrna) encoded by the gene, the amount or rate of synthesis of a polypeptide or protein encoded by the gene, or the amount or rate of synthesis of a biomolecule accumulated in a cell or biological fluid. The term "expression level" refers to the absolute amount of a molecule or the relative amount of a molecule in a sample, as determined under steady-state or non-steady-state conditions.

The mRNA that is "up-regulated" is typically increased under a given treatment or condition or in certain patient groups. An mRNA that is "down-regulated" generally refers to a decrease in the expression level of the mRNA in response to a given treatment or condition, or in certain patient groups. In some cases, mRNA levels may remain unchanged under a given treatment or condition. mRNA from patient samples is "up-regulated" upon treatment with the drug, as compared to untreated controls. Such upregulation can be, for example, an increase of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 500%, about 1,000%, about 5,000%, or more, compared to a control mRNA level. Alternatively, mRNA may be "down-regulated" or expressed at lower levels in response to administration of certain compounds or other agents. The downregulated mRNA can be, for example, present at a level that is about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 1%, or less of the level of a comparative control mRNA.

Similarly, the level of a polypeptide or protein biomarker from a patient sample may be increased when treated with a drug compared to an untreated control. Such an increase can be about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 500%, about 1,000%, about 5,000%, or more of the level of the comparative control protein. Alternatively, the level of protein biomarkers may be decreased in response to administration of certain compounds or other agents. Such a reduction can be present, for example, at a level of about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 1%, or less of the level of the comparative control protein.

As used herein, the terms "determining," "measuring," "evaluating," "assessing," and "determining" generally refer to any form of measurement and include determining whether an element is present. These terms include quantitative and/or qualitative determinations. The evaluation may be relative or absolute. The "evaluating the presence" may include determining the number of things present and determining whether it is present.

The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe a polymer of any length composed of nucleotides, such as deoxyribonucleotides or ribonucleotides, or a synthetically produced compound that can hybridize to two naturally occurring nucleic acids in a sequence-specific manner similar to that of the naturally occurring nucleic acids, e.g., can participate in Watson-Crick base-pairing interactions. As used herein in the context of polynucleotide sequences, the term "plurality of bases" (or "one base") is synonymous with "plurality of nucleotides" (or "one nucleotide"), i.e., a monomeric subunit of a polynucleotide. The terms "nucleoside" and "nucleotide" are intended to include those moieties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses, or other heterocycles. In addition, the terms "nucleoside" and "nucleotide" include those moieties that contain not only conventional ribose and deoxyribose sugars, but also other sugars. Modified nucleosides or nucleotides also include modifications on the sugar moiety, for example, wherein one or more hydroxyl groups are replaced with halogen atoms or aliphatic groups or are functionalized as ethers, amines, or the like. "analog" refers to a molecule having a structural feature that is considered in the literature to be a mimetic, derivative, having a similar structure, or other similar term, and includes, for example, polynucleotides that incorporate non-natural nucleotides, nucleotide mimetics such as 2' -modified nucleosides, peptide nucleic acids, oligonucleotide phosphonates, and any polynucleotide to which substituents have been added, such as protecting groups or linking moieties.

The term "complementary" refers to specific binding between polynucleotides based on polynucleotide sequences. As used herein, a first polynucleotide and a second polynucleotide are complementary if they bind to each other in a hybridization assay under stringent conditions, e.g., if they produce a given or detectable level of signal in the hybridization assay. If portions of the polynucleotide follow conventional base pairing rules, e.g., A pairs with T (or U) and G pairs with C, they are complementary to each other, although there may be small regions (e.g., less than about 3 bases) of mismatched, inserted, or deleted sequences.

The terms "isolated" and "purified" refer to the separation of a substance (such as mRNA, DNA, or protein) such that the substance constitutes a significant portion of the sample in which it resides, i.e., greater than the portion of the substance typically found in its native or non-isolated state. Typically, a significant portion of the sample constitutes, for example, greater than 1%, greater than 2%, greater than 5%, greater than 10%, greater than 20%, greater than 50% or more of the sample, often up to about 90-100%. For example, a sample of isolated mRNA may typically comprise at least about 1% total mRNA. Techniques for purifying polynucleotides are well known in the art and include, for example, gel electrophoresis, ion exchange chromatography, affinity chromatography, flow sorting, and sedimentation by density.

As used herein, the term "coupled" indicates a direct or indirect attachment. In the context of chemical structures, "associated with" (or "bonded to") may refer to the presence of a chemical bond that directly connects two moieties or indirectly connects two moieties (e.g., via a linking group or any other intervening moiety of a molecule). The chemical bonds may be covalent bonds, ionic bonds, coordination complexes, hydrogen bonds, van der waals interactions, or hydrophobic packing, or may exhibit characteristics of multiple types of chemical bonds. In certain instances, "coupled" includes embodiments that are directly attached and embodiments that are indirectly attached.

As used herein, the term "sample" relates to a material or mixture of materials, typically (but not necessarily) in fluid form, containing one or more components of interest.

As used herein, "biological sample" refers to a sample obtained from a biological subject, including samples of biological tissue or fluid origin obtained, arrived at, or collected in vivo or in situ. Biological samples also include samples from a region of a biological subject containing pre-cancerous or cancerous cells or tissues. Such samples may be, but are not limited to, organs, tissues and cells isolated from mammals. Exemplary biological samples include, but are not limited to, cell lysates, cells, tissues, organs, organelles, biological fluids, blood samples, urine samples, skin samples, and the like. Preferred biological samples include, but are not limited to, whole blood, partially purified blood, PBMCs, tissue biopsies (including tumor biopsies), circulating tumor cells, and the like.

As used herein, the term "polymerase chain reaction" or "PCR" generally refers to a procedure in which small amounts of nucleic acid, RNA, and/or DNA are amplified, as described, for example, in U.S. patent No. 4,683,195. In general, it is desirable to have available sequence information from the ends of the region of interest or beyond so that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to the opposite strand of the template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, phage or plasmid sequences, and the like. See generally Mullis et al, Cold Spring Harbor Symp. Quant. biol.1987,51: 263-273;PCR Technology(Stockton Press, NY, Erlich eds, 1989).

As used herein, "tautomer" refers to isomeric forms of a compound that are in equilibrium with each other. The concentration of the isomeric forms will depend on the environment in which the compound is found and may vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution. For example, in aqueous solution, pyrazoles may exhibit the following isomeric forms, which are referred to as tautomers of each other:

As used herein and unless otherwise indicated, the term "pharmaceutically acceptable salt" encompasses non-toxic acid and base addition salts of the compounds to which the term refers. Acceptable non-toxic acid addition salts include those derived from organic and inorganic acids known in the art, including, for example, hydrochloric, hydrobromic, phosphoric, sulfuric, methanesulfonic, acetic, tartaric, lactic, succinic, citric, malic, maleic, sorbic, aconitic, salicylic, phthalic, embolic, heptanoic, and the like. Compounds that are acidic in nature are capable of forming salts with various pharmaceutically acceptable bases. The bases which can be used for the preparation of the pharmaceutically acceptable base addition salts of such acidic compounds are those which form non-toxic base addition salts, i.e. salts containing a pharmacologically acceptable cation, such as, but not limited to, alkali metal or alkaline earth metal salts (in particular calcium, magnesium, sodium or potassium salts). Suitable organic bases include, but are not limited to, N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), lysine, and procaine.

As used herein and unless otherwise indicated, the term "solvate" means a compound provided herein or a salt thereof, which further includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. In the case where the solvent is water, the solvate is a hydrate.

As used herein and unless otherwise indicated, the term "co-crystal" means a crystalline form containing more than one compound in the crystal lattice. A co-crystal comprises a crystalline molecular complex of two or more non-volatile compounds bonded together by non-ionic interactions in a crystal lattice. As used herein, co-crystals include pharmaceutical co-crystals, wherein a crystalline molecular complex contains a therapeutic compound and one or more additional non-volatile compounds (referred to herein as one or more anti-molecules). The counter molecule in the pharmaceutical co-crystal is typically a non-toxic pharmaceutically acceptable molecule, such as, for example, a food additive, a preservative, a pharmaceutical excipient, or other Active Pharmaceutical Ingredient (API). In some embodiments, the pharmaceutical co-crystal enhances certain physicochemical properties (e.g., solubility, dissolution rate, bioavailability, and/or stability) of the drug product without compromising the chemical structural integrity of the API. See, e.g., Jones et al, MRS Bulletin 2006,31,875- & 879; trask, mol. pharmaceuticals 2007,4(3): 301-309; schultheiss and Newman, Crystal Growth & Design 2009,9(6): 2950-; shan and Zaworkko, Drug Discovery Today 2008,13(9/10): 440-446; and Vishweshwar et al, J.Pharm.Sci.2006,95(3): 499-.

As used herein and unless otherwise indicated, the term "stereoisomer" encompasses all enantiomerically/stereomerically pure and enantiomerically/stereomerically enriched compounds of the invention.

As used herein and unless otherwise indicated, the term "stereomerically pure" means a composition that comprises one stereoisomer of a compound and is substantially free of other stereoisomers of the compound. For example, a stereomerically pure composition of a compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure composition of a compound having two chiral centers will be substantially free of other diastereomers of the compound. Typical stereoisomerically pure compounds comprise greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, more preferably greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, even more preferably greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, and most preferably greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound.

As used herein and unless otherwise indicated, the term "stereomerically enriched" means a composition comprising greater than about 60% by weight of one stereoisomer of a compound, preferably greater than about 70% by weight, more preferably greater than about 80% by weight of one stereoisomer of a compound. As used herein and unless otherwise indicated, the term "enantiomerically pure" means a stereomerically pure composition of a compound having one chiral center. Similarly, the term "stereomerically enriched" means a stereomerically enriched composition of compounds having one chiral center.

As used herein and unless otherwise indicated, the term "prodrug" means a derivative of a compound that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide the compound. Examples of prodrugs include, but are not limited to, derivatives of the compounds described herein (e.g., compound 1) that comprise biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogs.

It should also be noted that compounds may contain unnatural proportions of atomic isotopes at one or more atoms. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (A), (B), (C), (D) and D) an) a) to ³ H) Iodine-125 (1) ¹²⁵ I) Sulfur-35 (a) ³⁵ S) or carbon-14 ( ¹⁴ C) Or may be isotopically enriched, such as enriched with deuterium (A), (B), (C), (D) and D) in a ² H) Carbon-13 (C) ¹³ C) Or nitrogen-15 ( ¹⁵ N). As used herein, an "isotopologue" is an isotopically enriched compound. The term "isotopically enriched" refers to atoms having an isotopic composition different from the natural isotopic composition of the atoms. "isotopically enriched" can also refer to compounds containing at least one atom having an isotopic composition different from the natural isotopic composition of the atom. The term "isotopic composition" refers to the amount of each isotope present for a given atom. Radiolabeled and isotopically enriched compounds are useful as therapeutic agents, for example, cancer and inflammation therapeutic agents; research reagents, such as binding assay reagents; and diagnostic agents, such as in vivo imaging agents. All isotopic variations of the compounds as described herein, whether radioactive or not, are intended to be encompassed within the scope of the embodiments provided herein. In some embodiments, isotopologues of the compounds are provided, e.g., isotopologues are deuterium, carbon-13, or nitrogen-15 enriched compounds. In some embodiments, isotopologues provided herein are deuterium enriched compounds. In some embodiments, isotopologues provided herein are deuterium enriched compounds, wherein deuteration occurs at a chiral center. In some embodiments, provided herein are isotopologues of the compounds provided herein, wherein deuteration occurs at a chiral center. In some embodiments, provided herein are isotopologues of compound D, wherein the deuteration occurs at a chiral center.

The term "about" or "approximately" means an acceptable error for a particular value as determined by one of ordinary skill in the art, depending in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

It should be noted that if there is a discrepancy between the structure being depicted and the name given to that structure, the structure being depicted will be given greater weight. Additionally, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of it.

The practice of the embodiments provided herein will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. Examples of particularly suitable text for review are as follows: the result of Sambrook et al, Molecular Cloning:A Laboratory Manual(4 th edition 2014); the edition by Glover (r) is as follows,DNA Cloningvolumes I and II (1995, 2 nd edition);Immunochemical Methods in Cell and Molecular Biology(Academic Press, London); the scope of the crops is shown in the specification,Protein Purification:Principles and Practice(Springer Verlag, New York, 3 rd edition 1993); and the Weir and Blackwell editions,Handbook of Experimental Immunologyvolumes I-IV (5 th edition 1996).

5.2. Gene sets, biomarkers, and methods of use thereof

5.2.1 Gene set

The methods provided herein are based in part on the following findings: a detectable increase in the expression level of certain gene sets (or gene signatures) is observed in a subject with cancer (e.g., a hematological cancer, such as lymphoma, MM, or leukemia) responsive to a given treatment (e.g., a compound, such as compound D, or a stereoisomer or a mixture of stereoisomers, a tautomer, a pharmaceutically acceptable salt, a solvate, an isotopologue, a prodrug, a hydrate, a co-crystal, a clathrate, or a polymorph thereof); and the expression levels of the gene set can be used to predict the responsiveness of the subject to treatment. In some embodiments, the compound is as described in section 5.5 herein. In one embodiment, the compound is compound D.

In certain embodiments, a gene set is a gene signature comprising a plurality of genes that are related by their association with certain cell types, biological functions, phenotypes, or cellular pathways, and the like. For example, in one particular embodiment, the genes within a gene signature are related by their association with a stem cell or a subpopulation of stem cells (e.g., LSCs).

In some embodiments, the genetic signature comprises at least one gene selected from the group of genes through which the set of genes is associated with a certain cell type, biological function, cellular pathway, or the like. In other embodiments, the characteristic comprises two, three, four, five, six, seven, eight, nine, ten, twenty, thirty, forty, fifty, or all genes selected from a group of related genes.

In one aspect, provided herein is a method of identifying a subject having cancer who is likely to respond to a treatment comprising a compound provided herein, or predicting the responsiveness of a subject having or suspected of having cancer to a treatment comprising the compound, the method comprising: i. providing a sample from the subject; measuring the gene expression level of one or more genes in the sample; calculating a Leukemia Stem Cell (LSC) signature score for the sample based on the gene expression level of the one or more genes; identifying the subject as likely to be responsive to a treatment comprising the compound if the level of the LSC signature score is above its reference level, wherein the therapeutic compound is compound D or a stereoisomer or mixture of stereoisomers thereof, an isotopologue, a pharmaceutically acceptable salt, a tautomer, a solvate, a hydrate, a co-crystal, a clathrate, or a polymorph thereof.

In some embodiments, provided herein is a panel of methods of treating a subject having cancer with a compound, the method comprising identifying a subject having cancer who is likely to respond to a treatment comprising the compound using a method provided herein (e.g., described above), and administering a therapeutically effective amount of the compound to the subject if the subject is identified as likely to respond to a treatment comprising the compound.

In certain embodiments, the cancer is a hematologic cancer. In one embodiment, the hematologic cancer is lymphoma. In another embodiment, the hematologic cancer is leukemia. In yet another embodiment, the hematologic cancer is MM. In a specific embodiment, the leukemia is ALL. In another specific embodiment, the leukemia is AML. In yet another specific embodiment, the leukemia is CLL. In yet another embodiment, the leukemia is CML.

In some embodiments, the AML is relapsed. In certain embodiments, the AML is refractory. In other embodiments, the AML is resistant to conventional therapies.

In a specific embodiment, provided herein is a method of identifying a subject having AML or predicting the responsiveness of a subject having or suspected of having AML to a treatment comprising a compound provided herein that is likely to respond to the treatment comprising the compound, the method comprising: i. providing a sample from the subject; measuring the gene expression level of one or more genes in the sample; calculating a Leukemia Stem Cell (LSC) signature score for the sample based on the gene expression level of the one or more genes; identifying the subject as likely to be responsive to a treatment comprising the compound if the level of the LSC signature score is above its reference level, wherein the therapeutic compound is compound D or a stereoisomer or mixture of stereoisomers thereof, an isotopologue, a pharmaceutically acceptable salt, a tautomer, a solvate, a hydrate, a co-crystal, a clathrate, or a polymorph thereof.

In certain embodiments, the methods provided herein are methods of identifying a subject with cancer who is likely to be responsive to a therapeutic compound. In some embodiments, the methods provided herein are methods of predicting the responsiveness of a subject having or suspected of having cancer to a therapeutic compound. In other embodiments, the methods provided herein are methods of treating cancer with a therapeutic compound. In yet other embodiments, the cancer is characterized by an increased level of LSC signature (or higher LSC signature score). In still other embodiments, the LSC feature is an LSC feature described herein. In one embodiment, provided herein is a method of treating cancer characterized by increasing the levels of LSC signature described herein (or higher LSC signature scores) with a therapeutic compound. In another embodiment, provided herein is a method of treating leukemia characterized by increasing the level of a characteristic LSC (or higher characteristic LSC score) described herein with a therapeutic compound. In yet another embodiment, provided herein is a method of treating AML characterized by increasing the level of a characteristic LSC (or higher characteristic LSC score) described herein with a therapeutic compound.

In certain embodiments of the methods provided herein, the reference level (reference level of LSC feature score) is the LSC feature level (or LSC feature score) in the control. In some embodiments, the control is obtained from a healthy subject not suffering from cancer. In other embodiments, the control is obtained from a subject having cancer but a good prognostic risk. In yet other embodiments, the control is obtained from a subject having cancer, and the cancer has been ameliorated or cured by a treatment other than administration of a therapeutic compound described herein to the subject. In other embodiments, the control is obtained from a subject who has cancer but is not responsive to the therapeutic compound. In yet other embodiments, the control is from the same tissue or cell source as the sample (e.g., blood or certain blood cells). In yet other embodiments, the control is a cell line (e.g., an AML cell line). In one embodiment, the reference level is the level of an LSC signature in a control obtained from a healthy subject without cancer, and the control is from the same tissue or cellular source as the sample (e.g., blood or certain blood cells). In another embodiment, the reference level is the level of an LSC signature in a control obtained from a subject having cancer but a good prognostic risk, and the control is from the same tissue or cellular source as the sample (e.g., blood or certain blood cells). In yet another embodiment, the reference level is a level of an LSC signature in a control obtained from a subject having cancer, and the cancer has been ameliorated or cured by a treatment other than administering to the subject a therapeutic compound described herein, and the control is from the same tissue or cellular source as the sample (e.g., blood or certain blood cells). In yet another embodiment, the reference level is a level of an LSC characteristic in a control obtained from a subject having cancer but not responding to the therapeutic compound, and the control is from the same tissue or cellular source as the sample (e.g., blood or certain blood cells). In yet another embodiment, the reference level is a level of an LSC signature in a control as a cell line. In yet another embodiment, the reference level is a level of an LSC signature in a control cell line derived from the same cellular source as the cancer (e.g., white blood cells, embryonic cells, etc.). In one embodiment, the reference level is a level of an LSC signature in a control that is a cancer cell line. In another embodiment, the reference level is a level characteristic of LSC in a control as an AML cell line. In yet another embodiment, the reference level (or reference score for the LSC feature) is determined based on LSC feature scores obtained from the population. In some embodiments, the reference score for the LSC feature is predetermined.

In some embodiments, the subject has received prior treatment prior to the methods provided herein. In certain embodiments, the prior treatment is a treatment other than administering to the subject the same therapeutic compound as the methods provided herein. In other embodiments, the prior treatment is administration of the same therapeutic compound to the subject as the methods provided herein. In one embodiment, the prior treatment comprises the same therapeutic compound with the same dosing regimen as the methods provided herein. In another embodiment, the prior treatment comprises the same therapeutic compound with a different dosing regimen (e.g., different amounts of therapeutic compound and/or frequency of administration) as compared to the methods provided herein. In some embodiments where the subject received prior treatment prior to the methods provided herein, the control is obtained from the same subject prior to the prior treatment. In particular embodiments, the control is from the same tissue or cell source (e.g., blood or certain blood cells) as the sample prior to the prior treatment. In some embodiments, the prior treatment is one or more agents selected from daunomycin, cytarabine (ara-C), and gemtuzumab ozogamicin or is resistant to chemotherapy.

In certain embodiments, the gene set comprises gene signatures associated with certain cell types (e.g., stem cells). In some embodiments, the gene set comprises gene signatures associated with certain biological functions (e.g., protein metabolism). In other embodiments, the gene set comprises gene signatures associated with certain cellular pathways (e.g., UPR pathways). In one embodiment, the gene set comprises gene signatures associated with Leukemic Stem Cells (LSCs). In another embodiment, the gene set comprises LSC gene signatures.

In some embodiments, the LSC gene signature comprises one or more genes selected from the group consisting of: CD34, SPINK2, LAPTM48, HOXA5, GUCY1A3, SHANK3, ANGPT1, ARHGAP22, LOC284422, MYCN, MAMDC2, PRSSL1, KIAA0125, GPSM1, HOXA9, MMRN1, FSCN1, DNMT38, HOXA6, AIF1L, SOCS2, CDK6, FAM69B, NGFRAP1, C3orf54, CPXM1, TNFRSF4, ZBTB46, DPYSL3, NYLNRIN, COL 1, FAM30A, C10orf140, SPNS A, GPR 4, AKR1C A, FLT 4, TFPI 685I, KCNK A, ADMP A, 685A, 685A, 685 FCS A, 685 FCS A, 685 FCS A, 685 FCS A, 685 FCS A, 685 FCS A, 685 FCS A, 685 and A, 685 FCS A, 685 and A, 685 FCS A, 685 and A, 685.

In other embodiments, the LSC gene signature comprises one or more genes selected from the group consisting of: CD34, SPINK2, LAPTM48, HOXA5, GUCY1A3, SHANK3, ANGPT1, ARHGAP22, LOC284422, MYCN, MAMDC2, PRSSL1, KIAA0125, GPSM1, HOXA9, MMRN1, FSCN1, DNMT38, HOXA6, AIF1L, SOCS2, CDK6, FAM69B, NGFRAP1, C3orf54, CPXM1, TNFRSF4, ZBTB4, DPYSL 4, NYRRIN, COL24A 4, FAM30 4, C10orf140, SPNS 4, GPR 4, AKR1C 4, FLT 4, TFPI 4, KCR 4, C150, VWF 4, ATP 4,

ATP

4, 4 and AFVM 4.

In certain embodiments, the LSC signature comprises at least one gene selected from table 1.

Table 1: LSC17 characteristic

AKR1C3
	ARHGAP22
CD34
	CDK6
CPXM1
	DNMT3B
DPYSL3
	EMP1
GPR56
	KIAA0125
LAPTM4B
	MMRN1
NGFRAP1
	NYNRIN
SMIM24
	SOCS2
ZBTB46

In certain embodiments, the LSC signature comprises at least one gene selected from the group consisting of: AKR1C3, ARHGAP22, CD34, CDK6, CPXM1, DNMT3B, DPYSL3, EMP1, GPR56, KIAA0125, lamm 4B, MMRN1, NGFRAP1, NYNRIN, SMIM24, SOCS2, and ZBTB 46. In one embodiment, the LSC signature comprises AKR1C 3. In one embodiment, the LSC feature comprises ARHGAP 22. In another embodiment, the LSC signature comprises CD 34. In yet another embodiment, the LSC characteristic comprises CDK 6. In yet another embodiment, the LSC feature comprises CPXM 1. In one embodiment, the LSC feature comprises DNMT 3B. In another embodiment, the LSC feature comprises DPYSL 3. In yet another embodiment, the LSC signature comprises EMP 1. In yet another embodiment, the LSC signature comprises GPR 56. In one embodiment, the LSC signature comprises KIAA 0125. In another embodiment, the LSC characteristic comprises lamtm 4B. In yet another embodiment, the LSC feature comprises MMRN 1. In yet another embodiment, the LSC signature comprises NGFRAP 1. In one embodiment, the LSC feature comprises NYNRIN. In another embodiment, the LSC feature comprises SMIM 24. In yet another embodiment, the LSC feature comprises SOCS 2. In yet another embodiment, the LSC signature comprises ZBTB 46.

In certain embodiments, the LSC signature comprises two genes selected from table 1. In some embodiments, the LSC signature comprises three genes selected from table 1. In other embodiments, the LSC signature comprises four genes selected from table 1. In yet other embodiments, the LSC signature comprises five genes selected from table 1. In still other embodiments, the LSC signature comprises six genes selected from table 1. In certain embodiments, the LSC signature comprises seven genes selected from table 1. In some embodiments, the LSC signature comprises eight genes selected from table 1. In other embodiments, the LSC signature comprises nine genes selected from table 1. In yet other embodiments, the LSC signature comprises ten genes selected from table 1. In still other embodiments, the LSC signature comprises twelve genes selected from table 1. In certain embodiments, the LSC signature comprises fourteen genes selected from table 1. In some embodiments, the LSC signature comprises sixteen genes selected from table 1. In other embodiments, the LSC signature comprises all seventeen genes selected from table 1, which are referred to as "LSC 17" or "LSC 17 signature".

In some embodiments, the LSC feature score (LSC17 score) is calculated as follows: (weight of expression level of DNMT 3B. times. DNMTT 3B) + (weight of expression level of ZBTB 46. times. ZBTB 46) + (weight of expression level of NYNRIN. times. NYNRIN) + (weight of expression level of ARHGAP 22. times. ARHGAP 22) + (weight of expression level of LAPTM 4B. times. LAPTM 4B) + (weight of expression level of MMRN 1. times. MMRN 1) + (weight of expression level of DPYSL 1. times. DPYSL 1) + (weight of expression level of KIAA 0125. times. KIAA 0125) + (weight of expression level of CDK 1. times. CDK 1) + (weight of CPXM 1. times. weight of SOCS 1) + (weight of expression level of SMIM 1. times. SMIM 1. times. weight of DSM 1) + (weight of EMXM 1. times. EMP 1. times. 1. times. weight of SOCS 1) + (weight of GPR 1. times. of GPR 1) + (weight of GPR 1. times. of GPR 1) + (weight of GPR 1); and DNMTT3B is in the range of 0.06 to 0.1, ZBTB46 is in the range of-0.05 to-0.01, NYNRIN is in the range of-0.01 to 0.03, ARHGAP22 is in the range of-0.03 to 0.01, lamm 4B is in the range of-0.015 to 0.025, MMRN1 is in the range of 0.005 to 0.045, DPYSL3 is in the range of 0.01 to 0.05, KIAA0125 is in the range of 0.009 to 0.039, CDK6 is in the range of-0.040.05, CPXM1 is in the range of-0.045 to 0.005, SOCS2 is in the range of 0.06007 to 0.007, smcs 632 is in the range of 0.040.047, emm to 0.040.05, CPXM1 is in the range of-0.035 to 0.005, SOCS 462 is in the range of 0.007 to 0.007, emm 0.380.05 is in the range of emtb 3 to 0.05, GPR is in the range of 0.05, and emr 3 is in the range of 0.05 to 0.05, and emr 0.02 to 0.05 is in the range of emr 3, and emr 0.05 to 0.05, and emtb 6319 to 0.05 is in the range of emr 3 to 0.05, and emr 0.05, and emtb 6319 to 0.05 is in the range of emr 3 to 0.05.

In some embodiments, DNMTT3B is weighted in the range of 0.08 to 0.09, ZBTB46 is weighted in the range of-0.03 to-0.04, NYNRIN is weighted in the range of-0.008 to 0.009, ARHGAP22 is weighted in the range of-0.015 to 0.01, lamtm 4B is weighted in the range of-0.006 to 0.005, MMRN1 is weighted in the range of 0.02 to 0.03, DPYSL3 is weighted in the range of 0.02 to 0.03, KIAA0125 is weighted in the range of 0.01 to 0.02, CDK6 is weighted in the range of-0.08 to-0.07, CPXM1 is weighted in the range of-0.02 to-0.03, SOCS2 is weighted in the range of 0.02 to 0.03, smip 460.04 is weighted in the range of-0.04 to 0.05, and GPR 0.04 is weighted in the range of 0.04 to 0.05, emr 3 to 0.05, and GPR is weighted in the range of 0.04 to 0.05.

In some embodiments, DNMT3B is weighted about 0.0874, ZBTB46 is weighted about-0.0347, NYNRIN is weighted about 0.00865, ARHGAP22 is weighted about-0.0138, lamm 4B is weighted about 0.00582, MMRN1 is weighted about 0.0258, DPYSL3 is weighted about 0.0284, KIAA0125 is weighted about 0.0196, CDK6 is weighted about-0.0704, CPXM1 is weighted about-0.0258, SOCS2 is weighted about 0.0271, SMIM24 is weighted about-0.0226, EMP1 is weighted about 0.0146, NGFRAP1 is weighted about 0.0465, CD34 is weighted about CD 86 0.0338, AKR1C3 is weighted about-38 0.0402, and GPR56 is weighted about 0.0501.

In a particular embodiment, the LSC feature score (LSC17 score) is calculated as follows: (expression level of DNMT3B × 0.0874) + (expression level of ZBTB46 × -0.0347) + (NYNRIN × 0.00865) + (ARHGAP22 × -0.0138) + (lamm 4B expression level × 0.00582) + (MMRN1 × 0.0258) + (DPYSL3 expression level × 0.0284) + (KIAA0125 expression level × 0.0196) + (CDK6 expression level of × -0.0704) + (CPXM1 expression level × -0.0258) + (SOCS2 × 0.0271) + (SMIM24 expression level × -0.0226) + (EMP1 expression level × 0.0146) + (NGFRAP1 × 0.0465) + (CD34 expression level × 0.0338) + (AKR1 × 3) + (akgpr × 3646) + (akgpr 3646).

In some embodiments, the reference level is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8.0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.

In certain embodiments, the LSC signature comprises at least one gene selected from TNFRSF4, SLC4a1, SLC7a7, and AIM 2. In one embodiment, the LSC signature comprises TNFRSF 4. In one implementation, the LSC feature comprises SLC4a 1. In another embodiment, the LSC signature comprises SLC7a 7. In yet another embodiment, the LSC feature comprises AIM 2.

In certain embodiments, the LSC signature comprises two genes selected from TNFRSF4, SLC4a1, SLC7a7, and AIM 2. In some embodiments, the LSC signature comprises three genes selected from TNFRSF4, SLC4a1, SLC7a7, and AIM 2. In some embodiments, the LSC signature consists of TNFRSF4, SLC4a1, SLC7a7, and AIM2, which are referred to as LSC4 or LSC4 signatures.

In some embodiments, the LSC feature score (LSC4 feature score) is calculated as follows: (expression level of TNFRSF4 × weight of TNFRSF 4) + (expression level of SLC4a1 × weight of SLC4a 1) + (expression level of SLC7a7 × weight of SLC7a 7) + (expression level of AIM2 × weight of AIM 2); and wherein TNFRSF4 is weighted in the range of-2 to-1, SLC4A1 is weighted in the range of 11 to 15, SLC7A7 is weighted in the range of-5.5 to-1.5, and AIM2 is weighted in the range of-5 to-1.

In some embodiments, TNFRSF4 is weighted in the range of-1.5 to-1, SLC4a1 is weighted in the range of 13 to 14, SLC7a7 is weighted in the range of-4 to-3, and AIM2 is weighted in the range of-3 to-4.

In some embodiments, TNFRSF4 is weighted about-1.13, SLC4a1 is weighted about 13.59, SLC7a7 is weighted about-3.57, and AIM2 is weighted about-3.04.

In a particular embodiment, the LSC feature score is calculated as follows: (expression level of TNFRSF4 × -1.13) + (expression level of SLC4A1 × 13.59) + (expression level of SLC7A7 × -3.57) + (expression level of AIM2 × -3.04).

In certain embodiments, the LSC signature comprises at least one gene selected from SLC4a1, SLC7a7, and AIM 2. In certain embodiments, the LSC signature comprises two genes selected from SLC4a1, SLC7a7, and AIM 2. In some embodiments, the LSC signature consists of SLC4a1, SLC7a7, and AIM2, which are referred to as LSC3 or LSC3 signatures.

In some embodiments, the LSC feature score (LSC3 feature score) is calculated as follows: (SLC4a1 expression level x weight of SLC4a 1) + (SLC7a7 expression level x weight of SLC7a 7) + (AIM2 expression level x AIM2 weight); and wherein SLC4A1 is weighted in the range of 11 to 15, SLC7A7 is weighted in the range of-5.5 to-1.5, and AIM2 is weighted in the range of-5 to-1.

In some embodiments, SLC4a1 is weighted in the range of 13 to 14, SLC7a7 is weighted in the range of-4 to-3, and AIM2 is weighted in the range of-3 to-4.

In some embodiments, the SLC4a1 is weighted about 13.59, the SLC7a7 is weighted about-3.57, and the AIM2 is weighted about-3.04.

In a particular embodiment, the LSC feature score is calculated as follows: (SLC4A1 expression level X13.59) + (SLC7A7 expression level X-3.57) + (AIM2 expression level X-3.04).

In some embodiments, the methods provided herein comprise determining that the patient is likely to be responsive to a treatment comprising a compound provided herein if the LSC characteristic score in the sample is about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, or about 100-fold higher than the LSC characteristic reference score. In some embodiments, the LSC feature score is a LSC17 feature score. In some embodiments, the LSC feature score is a LSC4 feature score. In other embodiments, the LSC feature score is a LSC3 feature score.

5.2.2 cell surface markers

As shown below in section 6, treatment with a compound of the invention (e.g., compound D) induces a decrease or increase in certain cell types with certain cellular markers. For example, the proportion of blasts and/or the proportion of differentiated leukemic cells is altered after treatment with compound D. Thus, in another aspect, provided herein is a method of predicting responsiveness to a therapeutic compound (e.g., compound D, or a stereoisomer or a mixture of stereoisomers, a tautomer, a pharmaceutically acceptable salt, a solvate, an isotopologue, a prodrug, a hydrate, a co-crystal, a clathrate, or a polymorph thereof) based on a decrease or increase in certain cell types or associated cell surface markers.

In some embodiments, provided herein is a method of identifying a subject having cancer who is likely to respond to a treatment comprising a compound, or predicting the responsiveness of a subject having or suspected of having cancer to a treatment comprising the compound, the method comprising: i. providing a sample from the subject; administering the compound to the sample; measuring the proportion of one or more cell types; identifying the subject as likely to be responsive to a treatment comprising the compound if the ratio of the one or more cell types is different from the reference ratio of the cells, wherein the treatment compound is 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide having the structure (compound D):

In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of the compound if the subject is identified as likely to be responsive to a treatment comprising the compound.

In some embodiments, the reference proportion of a cell type is the proportion of the cell type in the sample prior to administration of the compound. In other embodiments, the reference ratio for a cell type is a predetermined ratio. In yet other embodiments, the reference proportion of a cell type is the proportion of said cell type in a sample obtained from a subject that is non-responsive to treatment with the compound.

In some embodiments, the method comprises measuring the proportion of primitive cells and/or the proportion of differentiated leukemia cells. In some embodiments, a decrease in the proportion of naive cells as compared to the proportion prior to administration of the compound indicates that the subject is likely to be responsive to a treatment comprising the compound. In other embodiments, an increase in the proportion of differentiated leukemia cells compared to the proportion prior to administration of the compound indicates that the subject is likely to be responsive to a treatment comprising the compound.

In some embodiments, the methods comprise measuring and comparing the proportion of CD34+, CD15+, CD14+ and/or CD11b + cells before and after administering the compound to the sample.

In some embodiments, the method comprises measuring the proportion of CD34+ cells, and wherein a decrease in the proportion of CD34+ cells as compared to the proportion of CD34+ cells prior to administration of the compound indicates that the subject is likely to be responsive to a treatment comprising the compound.

In other embodiments, the method comprises measuring the proportion of CD15+ cells and/or CD14+ cells, and wherein an increase in the proportion of CD15+ cells and/or CD14+ cells as compared to the proportion of CD15+ cells and/or CD14+ cells prior to administration of the compound indicates that the subject is likely to be responsive to a treatment comprising the compound.

In other embodiments, provided herein is a method of identifying a subject having cancer who is likely to respond to a treatment comprising a compound, or predicting the responsiveness of a subject having or suspected of having cancer to a treatment comprising the compound, the method comprising: i. providing a sample from the subject; administering the compound to the sample; measuring the level of one or more cell surface markers; identifying the subject as likely to respond to a treatment comprising the compound if the level of the one or more cell surface markers is different from a reference level, wherein the treatment compound is compound D or a stereoisomer or a mixture of stereoisomers thereof, an isotopologue, a pharmaceutically acceptable salt, a tautomer, a solvate, a hydrate, a co-crystal, a clathrate, or a polymorph thereof.

In some embodiments, the one or more cell surface markers are selected from the group consisting of CD34, CD15, CD14, and CD11 b.

In some embodiments, the methods comprise measuring the level of CD34 before and after administration of a compound (e.g., compound D), and a decrease in the level of CD34 after administration of the compound indicates that the subject is likely to be responsive to a treatment comprising the compound.

In other embodiments, the method comprises measuring the level of CD15 and/or CD14 before and after administration of the compound (e.g., compound D), and an increase in the level of CD15 and/or CD14 after administration of the compound indicates that the subject is likely to be responsive to a treatment comprising the compound.

Methods of determining the proportion of cell types having certain cell surface markers and methods of determining the level of cell surface markers in a sample are known in the art. An exemplary method is described in section 6 below.

In some embodiments, an increase means an increase of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a reference. In some embodiments, a decrease means a decrease of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a reference.

In certain embodiments, the cancer is a blood cancer. In one embodiment, the blood cancer is lymphoma. In another embodiment, the blood cancer is leukemia. In yet another embodiment, the blood cancer is MM. In a specific embodiment, the leukemia is ALL. In another specific embodiment, the leukemia is AML. In yet another specific embodiment, the leukemia is CLL. In yet another embodiment, the leukemia is CML.

In a specific embodiment, provided herein is a method of identifying a subject having AML who is likely to respond to a treatment comprising a compound provided herein or predicting responsiveness of a subject having or suspected of having AML to a treatment comprising the compound using the methods described above.

5.2.3 Selective treatment

In some embodiments of the various methods provided herein (including those described above), a compound provided herein is administered to a patient who has been determined to be likely to respond to the compound. Accordingly, in one aspect, provided herein is a method of selective treatment comprising administering a compound to a patient who has been determined to be likely to respond to the compound based on the methods described herein (including those described above).

In another particular embodiment, the compound is compound D or a stereoisomer or mixture of stereoisomers thereof, an isotopologue, a pharmaceutically acceptable salt, a tautomer, a solvate, a hydrate, a co-crystal, a clathrate, or a polymorph.

In some embodiments of the various methods provided herein, a therapeutic compound is administered to a patient who is likely to respond to the therapeutic compound. Also provided herein are methods of treating previously treated cancer in patients who have failed to respond to standard therapy, as well as previously untreated patients. The invention also encompasses methods of treating patients regardless of the age of the patient, although some diseases or disorders are more common in certain age groups. The invention further encompasses methods of treating patients who have undergone surgery in an attempt to treat the disease or disorder in question, as well as patients who have not undergone surgery. Since patients with cancer have heterogeneous clinical manifestations and different clinical outcomes, the treatment given to a patient may vary depending on his/her prognosis. A skilled clinician will be able to readily determine without undue experimentation the particular adjunctive agents, types of surgery and types of non-drug based standard therapies that may be effectively used to treat an individual patient suffering from cancer.

Administration and administration

In certain embodiments, a therapeutically or prophylactically effective amount of a compound provided herein. In certain embodiments, a therapeutically or prophylactically effective amount of compound D is from about 0.005 to about 20 mg/day, from about 0.05 to 20 mg/day, from about 0.01 to about 10 mg/day, from about 0.01 to about 7 mg/day, from about 0.01 to about 5 mg/day, from about 0.01 to about 3 mg/day, from about 0.05 to about 10 mg/day, from about 0.05 to about 7 mg/day, from about 0.05 to about 5 mg/day, from about 0.05 to about 3 mg/day, from about 0.1 to about 15 mg/day, from about 0.1 to about 10 mg/day, from about 0.1 to about 7 mg/day, from about 0.1 to about 5 mg/day, from about 0.1 to about 3 mg/day, from about 0.5 to about 10 mg/day, from about 0.05 to about 5 mg/day, from about 0.5 to about 3 mg/day, from about 0.5 to about 2 mg/day, from about 0.1 to about 3 mg/day, from about 3.8 mg/day, from about 3 mg/day, from about 0.1 to about 8 mg/day, from about 3 mg/day, from about 0.1 to about 10 mg/day, or from about 3 mg/day, From about 0.6 to about 10 mg/day or from about 0.6 to about 5 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.005 to about 20 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is about 0.05 to 20 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.01 to about 10 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.01 to about 7 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.01 to about 5 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.01 to about 3 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.05 to about 10 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.05 to about 7 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.05 to about 5 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.05 to about 3 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.1 to about 15 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.1 to about 10 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.1 to about 7 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.1 to about 5 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.1 to about 3 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.5 to about 10 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.5 to about 5 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.5 to about 3 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.5 to about 2 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.3 to about 10 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.3 to about 8.5 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.3 to about 8.1 mg/day. In one embodiment, a therapeutically or prophylactically effective amount of compound D is from about 0.6 to about 10 mg/day or from about 0.6 to about 5 mg/day.

In certain embodiments, the therapeutically or prophylactically effective amount is about 0.1, about 0.2, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 mg/day. In some such embodiments, the therapeutically or prophylactically effective amount is about 0.5, about 0.6, about 0.75, about 1, about 2, about 3, about 4, about 5, about 6, or about 7 mg/day. In some such embodiments, the therapeutically or prophylactically effective amount is about 0.6, about 1.2, about 1.8, about 2.4, or about 3.6 mg/day. In certain embodiments, a therapeutically or prophylactically effective amount is about 0.1 mg/day. In certain embodiments, a therapeutically or prophylactically effective amount is about 0.2 mg/day. In certain embodiments, a therapeutically or prophylactically effective amount is about 0.5 mg/day. In certain embodiments, a therapeutically or prophylactically effective amount is about 1 mg/day. In certain embodiments, a therapeutically or prophylactically effective amount is about 2 mg/day. In certain embodiments, a therapeutically or prophylactically effective amount is about 3 mg/day. In certain embodiments, a therapeutically or prophylactically effective amount is about 4 mg/day. In certain embodiments, a therapeutically or prophylactically effective amount is about 5 mg/day. In certain embodiments, a therapeutically or prophylactically effective amount is about 6 mg/day. In certain embodiments, a therapeutically or prophylactically effective amount is about 7 mg/day. In certain embodiments, a therapeutically or prophylactically effective amount is about 8 mg/day. In certain embodiments, a therapeutically or prophylactically effective amount is about 9 mg/day. In certain embodiments, a therapeutically or prophylactically effective amount is about 10 mg/day.

In one embodiment, the recommended daily dosage of compound D for the conditions described herein ranges from about 0.01mg to about 20mg per day, preferably given as a single once-a-day dose or as divided doses throughout the day. In one embodiment, the recommended daily dosage of compound D for the conditions described herein ranges from about 0.01mg to about 15mg per day, preferably given as a single once-a-day dose or as divided doses throughout the day. In one embodiment, the recommended daily dosage of compound D for the conditions described herein ranges from about 0.01mg to about 12mg per day, preferably given as a single once-a-day dose or as divided doses throughout the day. In some embodiments, the dose range is from about 0.1mg to about 10mg per day. In other embodiments, the dosage range is from about 0.5 to about 5 mg/day. Specific doses per day include 0.1, 0.2, 0.5, 0.6, 1, 1.2, 1.5, 1.8, 2, 2.4, 2.5, 3, 3.5, 3.6, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.4, 14.5, or 15 mg/day. In other embodiments, the dosage range is from about 0.5 to about 5 mg/day. Specific doses per day include 0.1, 0.2, 0.5, 0.6, 1, 1.2, 1.5, 1.8, 2, 2.4, 2.5, 3, 3.5, 3.6, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10mg per day. In one embodiment, the dose per day is 0.1 mg/day. In one embodiment, the dose per day is 0.2 mg/day. In one embodiment, the dose per day is 0.5 mg/day. In one embodiment, the dose per day is 0.6 mg/day. In one embodiment, the dose per day is 1mg per day. In one embodiment, the dose per day is 1.2 mg/day. In one embodiment, the dose per day is 1.5 mg/day. In one embodiment, the dose per day is 1.8 mg/day. In one embodiment, the dose per day is 2mg per day. In one embodiment, the dose per day is 2.4 mg/day. In one embodiment, the dose per day is 2.5 mg/day. In one embodiment, the dose per day is 3mg per day. In one embodiment, the dose per day is 3.5 mg/day. In one embodiment, the dose per day is 3.6 mg/day. In one embodiment, the dose per day is 4mg per day. In one embodiment, the dose per day is 4.5 mg/day. In one embodiment, the dose per day is 5mg per day. In one embodiment, the dose per day is 5.5 mg/day. In one embodiment, the dose per day is 6mg per day. In one embodiment, the dose per day is 6.5 mg/day. In one embodiment, the dose per day is 7mg per day. In one embodiment, the dose per day is 7.2 mg/day. In one embodiment, the dose per day is 7.5 mg/day. In one embodiment, the dose per day is 8 mg/day. In one embodiment, the dose per day is 8.5 mg/day. In one embodiment, the dose per day is 9mg per day. In one embodiment, the dose per day is 9.5 mg/day. In one embodiment, the dose per day is 10mg per day. In one embodiment, the dose per day is 12mg per day. In one embodiment, the dose per day is 10mg per day. In one embodiment, the dose per day is 12mg per day. In one embodiment, the dose per day is 14.4 mg/day. In one embodiment, the dose per day is 15 mg/day.

In a particular embodiment, the recommended starting dose may be 0.1, 0.5, 0.6, 0.7, 1, 1.2, 1.5, 1.8, 2, 2.4, 2.5, 3, 3.5, 3.6, 4, 4.5, 5, 5.5, 6, 6.5 or 7 mg/day. In another embodiment, the recommended starting dose may be 0.1, 0.5, 0.6, 1, 1.2, 1.8, 2, 2.4, 3, 3.6, 4, or 5 mg/day. In one embodiment, the dose may be escalated to 7, 8, 910, 12 or 15 mg/day. In one embodiment, the dosage may be escalated to 7, 8, 9, or 10 mg/day.

In a specific embodiment, compound D may be administered to a patient suffering from leukemia (including AML) in an amount of about 0.1 mg/day. In a particular embodiment, compound D may be administered to patients with leukemia (including AML) in an amount of about 1 mg/day. In a particular embodiment, compound D may be administered to patients with leukemia (including AML) in an amount of about 3 mg/day. In a particular embodiment, compound D may be administered to patients with leukemia (including AML) in an amount of about 4 mg/day. In a particular embodiment, compound D provided herein can be administered to a patient having leukemia (including AML) in an amount of about 5 mg/day. In a particular embodiment, compound D provided herein can be administered to a patient having leukemia (including AML) in an amount of about 6 mg/day. In a particular embodiment, compound D provided herein can be administered to a patient having leukemia (including AML) in an amount of about 7 mg/day. In a particular embodiment, compound D provided herein can be administered to a patient having leukemia (including AML) in an amount of about 10 mg/day. In a particular embodiment, compound D provided herein can be administered to a patient having leukemia (including AML) in an amount of about 12 mg/day. In a particular embodiment, compound D provided herein can be administered to a patient having leukemia (including AML) in an amount of about 15 mg/day.

In a specific embodiment, compound D can be administered to a subject with MDS in an amount of about 0.1 mg/day. In a particular embodiment, compound D can be administered to a subject with MDS in an amount of about 1 mg/day. In a particular embodiment, compound D can be administered to a subject with MDS in an amount of about 3 mg/day. In a particular embodiment, compound D can be administered to a subject with MDS in an amount of about 4 mg/day. In a particular embodiment, compound D provided herein can be administered to a subject with MDS in an amount of about 5 mg/day. In a particular embodiment, compound D provided herein can be administered to a patient with MDS in an amount of about 6 mg/day. In a particular embodiment, compound D provided herein can be administered to a patient with MDS in an amount of about 7 mg/day. In a particular embodiment, compound D provided herein can be administered to a patient with MDS in an amount of about 10 mg/day. In a particular embodiment, compound D provided herein can be administered to a patient with MDS in an amount of about 12 mg/day. In a particular embodiment, compound D provided herein can be administered to a subject with MDS in an amount of about 15 mg/day.

In certain embodiments, a therapeutically or prophylactically effective amount is from about 0.001 to about 20 mg/kg/day, from about 0.01 to about 15 mg/kg/day, from about 0.01 to about 10 mg/kg/day, from about 0.01 to about 9 mg/kg/day, from 0.01 to about 8 mg/kg/day, from about 0.01 to about 7 mg/kg/day, from about 0.01 to about 6 mg/kg/day, from about 0.01 to about 5 mg/kg/day, from about 0.01 to about 4 mg/kg/day, from about 0.01 to about 3 mg/kg/day, from about 0.01 to about 2 mg/kg/day, from about 0.01 to about 1 mg/kg/day, or from about 0.01 to about 0.05 mg/kg/day. In certain embodiments, a therapeutically or prophylactically effective amount is from about 0.001 to about 20 mg/kg/day. In certain embodiments, a therapeutically or prophylactically effective amount is from about 0.01 to about 15 mg/kg/day. In certain embodiments, a therapeutically or prophylactically effective amount is from about 0.01 to about 10 mg/kg/day. In certain embodiments, a therapeutically or prophylactically effective amount is from about 0.01 to about 9 mg/kg/day. In certain embodiments, a therapeutically or prophylactically effective amount is 0.01 to about 8 mg/kg/day. In certain embodiments, a therapeutically or prophylactically effective amount is from about 0.01 to about 7 mg/kg/day. In certain embodiments, a therapeutically or prophylactically effective amount is from about 0.01 to about 6 mg/kg/day. In certain embodiments, a therapeutically or prophylactically effective amount is from about 0.01 to about 5 mg/kg/day. In certain embodiments, a therapeutically or prophylactically effective amount is from about 0.01 to about 4 mg/kg/day. In certain embodiments, a therapeutically or prophylactically effective amount is from about 0.01 to about 3 mg/kg/day. In certain embodiments, a therapeutically or prophylactically effective amount is from about 0.01 to about 2 mg/kg/day. In certain embodiments, a therapeutically or prophylactically effective amount is from about 0.01 to about 1 mg/kg/day. In certain embodiments, a therapeutically or prophylactically effective amount is from about 0.01 to about 0.05 mg/kg/day.

The dosage administered may also be expressed in units other than mg/kg/day. For example, the dose administered parenterally may be expressed as mg/m ² The day is. One of ordinary skill in the art will readily know how to convert the dose from mg/kg/day to mg/m for a given subject's height or weight, or both ² Htm/day (see www.fda.gov/cd/cancer/animal frame). For example, for a 65kg human, a 1 mg/kg/day dose is approximately equal to 38mg/m ² The day is.

In certain embodiments, the amount of compound D administered is sufficient to provide a compound plasma concentration at steady state in the range of about 0.001 to about 500 μ M, about 0.002 to about 200 μ M, about 0.005 to about 100 μ M, about 0.01 to about 50 μ M, about 1 to about 50 μ M, about 0.02 to about 25 μ M, about 0.05 to about 20 μ M, about 0.1 to about 20 μ M, about 0.5 to about 20 μ M, or about 1 to about 20 μ M. In certain embodiments, the amount of compound D administered is sufficient to provide a compound plasma concentration at steady state in the range of about 0.001 to about 500 μ M, about 0.002 to about 200 μ M, about 0.005 to about 100 μ M, about 0.01 to about 50 μ M, about 1 to about 50 μ M, about 0.02 to about 25 μ M, about 0.05 to about 20 μ M, about 0.1 to about 20 μ M, about 0.5 to about 20 μ M, or about 1 to about 20 μ M.

In other embodiments, the amount of compound D formulation administered is sufficient to provide a compound plasma concentration at steady state in the range of about 5 to about 100nM, about 5 to about 50nM, about 10 to about 100nM, about 10 to about 50nM, or about 50 to about 100 nM. In other embodiments, the amount of compound D formulation administered is sufficient to provide a compound plasma concentration at steady state ranging from about 5 to about 100 nM. In other embodiments, the amount of compound D formulation applied is sufficient to provide a compound plasma concentration at steady state ranging from about 5 to about 50 nM. In other embodiments, the amount of compound D formulation administered is sufficient to provide a compound plasma concentration at steady state, ranging from about 10 to about 100 nM. In other embodiments, the amount of compound D formulation applied is sufficient to provide a compound plasma concentration at steady state ranging from about 10 to about 50 nM. In other embodiments, the amount of compound D formulation applied is sufficient to provide a compound plasma concentration at steady state ranging from about 50 to about 100 nM.

As used herein, the term "plasma concentration at steady state" is the concentration achieved after a period of time following administration of a formulation provided herein. Once steady state is reached, smaller peaks and troughs appear on the time-dependent curve of plasma concentration in solid form.

In certain embodiments, the amount of compound D formulation applied is sufficient to provide a maximum plasma concentration (peak concentration) of the compound in the range of about 0.001 to about 500 μ Μ, about 0.002 to about 200 μ Μ, about 0.005 to about 100 μ Μ, about 0.01 to about 50 μ Μ, about 1 to about 50 μ Μ, about 0.02 to about 25 μ Μ, about 0.05 to about 20 μ Μ, about 0.1 to about 20 μ Μ, about 0.5 to about 20 μ Μ or about 1 to about 20 μ Μ. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a maximum plasma concentration (peak concentration) of the compound in the range of about 0.001 to about 500 μ M. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a maximum plasma concentration (peak concentration) of the compound in the range of about 0.002 to about 200 μ M. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a maximum plasma concentration (peak concentration) of the compound in the range of about 0.005 to about 100 μ M. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a maximum plasma concentration (peak concentration) of the compound in the range of about 0.01 to about 50 μ M. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a maximum plasma concentration (peak concentration) of the compound in the range of about 1 to about 50 μ Μ. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a maximum plasma concentration (peak concentration) of the compound in the range of about 0.02 to about 25 μ M. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a maximum plasma concentration (peak concentration) of the compound in the range of about 0.05 to about 20 μ M. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a maximum plasma concentration (peak concentration) of the compound in the range of about 0.1 to about 20 μ M. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a maximum plasma concentration (peak concentration) of the compound in the range of about 0.5 to about 20 μ M. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a maximum plasma concentration (peak concentration) of the compound in the range of about 1 to about 20 μ Μ.

In certain embodiments, the amount of compound D formulation applied is sufficient to provide a minimum plasma concentration (trough concentration) of the compound in the range of about 0.001 to about 500 μ Μ, about 0.002 to about 200 μ Μ, about 0.005 to about 100 μ Μ, about 0.01 to about 50 μ Μ, about 1 to about 50 μ Μ, about 0.01 to about 25 μ Μ, about 0.01 to about 20 μ Μ, about 0.02 to about 20 μ Μ or about 0.01 to about 20 μ Μ. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a minimum plasma concentration (trough concentration) of the compound in the range of about 0.001 to about 500 μ M. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a minimum plasma concentration (trough concentration) of the compound in the range of about 0.002 to about 200 μ Μ. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a minimum plasma concentration (trough concentration) of the compound in the range of about 0.005 to about 100 μ Μ. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a minimum plasma concentration (trough concentration) of the compound in the range of about 0.01 to about 50 μ Μ. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a minimum plasma concentration (trough concentration) of the compound in the range of about 1 to about 50 μ Μ, about 0.01 to about 25 μ Μ. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a minimum plasma concentration (trough concentration) of the compound in the range of about 0.01 to about 20 μ Μ. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a minimum plasma concentration (trough concentration) of the compound in the range of about 0.02 to about 20 μ Μ. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a minimum plasma concentration (trough concentration) of the compound in the range of about 0.02 to about 20 μ Μ. In certain embodiments, the amount of compound D formulation applied is sufficient to provide a minimum plasma concentration (trough concentration) of the compound in the range of about 0.01 to about 20 μ Μ.

In certain embodiments, the amount of compound D formulation administered is sufficient to provide an area under the curve (AUC) of the compound in the range of about 100 to about 100,000ng hr/mL, about 1,000 to about 50,000ng hr/mL, about 5,000 to about 25,000ng hr/mL, or about 5,000 to about 10,000ng hr/mL. In certain embodiments, the amount of compound D formulation applied is sufficient to provide an area under the curve (AUC) of the compound in the range of about 100 to about 100,000ng hr/mL. In certain embodiments, the amount of compound D formulation applied is sufficient to provide an area under the curve (AUC) of the compound in the range of about 1,000 to about 50,000ng hr/mL. In certain embodiments, the amount of compound D formulation applied is sufficient to provide an area under the curve (AUC) of the compound in the range of about 5,000 to about 25,000ng hr/mL. In certain embodiments, the amount of compound D formulation applied is sufficient to provide an area under the curve (AUC) of the compound in the range of about 5,000 to about 10,000ng hr/mL.

In certain embodiments, a patient to be treated with one of the methods provided herein has not been treated with an anti-cancer therapy prior to administration of a compound D formulation provided herein. In certain embodiments, a patient to be treated with one of the methods provided herein has been treated with an anti-cancer therapy prior to administration of a compound D formulation provided herein. In certain embodiments, a patient to be treated with one of the methods provided herein has developed resistance to an anticancer therapy.

The methods provided herein encompass treating patients regardless of the age of the patient, although some diseases or disorders are more common in certain age groups.

The compound D formulations provided herein can be delivered as a single dose, such as, for example, a single bolus injection, or over time, such as, for example, a continuous infusion over time or a split bolus administration over time. If desired, compound D formulations can be repeatedly administered, e.g., until the patient experiences stable disease or regression or until the patient experiences disease progression or unacceptable toxicity. For example, for solid tumors, stable disease generally means that the vertical diameter of the measurable lesion is not increased by 25% or more compared to the last measurement. Response Evaluation Criteria In Solid Turbines (RECIST) Guidelines, Journal of the National Cancer Institute 92(3): 205-. Stable disease or lack thereof is determined by methods known in the art, such as evaluating patient symptoms; physical examination; tumor visualization using X-ray, CAT, PET or MRI scan imaging and other commonly accepted evaluation modalities.

Compound D formulations provided herein may be administered once daily (QD) or divided into multiple daily doses, such as twice daily (BID), three times daily (TID), and four times daily (QID). In addition, administration can be continuous (i.e., daily, for a number of consecutive days, or for each day), intermittent, such as on a periodic basis (i.e., including a rest period of days, weeks, or months without drug). As used herein, the term "daily" is intended to mean that the therapeutic compound is administered once or more than once per day, e.g., for a period of time. The term "continuous" is intended to mean daily administration of a therapeutic compound for an uninterrupted period of at least 10 days to 52 weeks. As used herein, the term "intermittent" or "intermittently" is intended to mean stopping and starting at regular or irregular intervals. For example, intermittent administration of a compound D formulation is one to six days per week, on a periodic basis (e.g., one to ten consecutive days of a 28-day cycle for daily administration followed by a rest period during which no administration occurs for the remainder of the 28-day cycle, or two to eight consecutive weeks for daily administration followed by a rest period during which no administration occurs for up to one week), or on alternate days. Periodic therapy with compound D is discussed elsewhere herein.

In some embodiments, the frequency of administration ranges from about a daily dose to about a monthly dose. In certain embodiments, the administration is once daily, twice daily, three times daily, four times daily, once every other day, twice weekly, once every two weeks, once every three weeks, or once every four weeks. In one embodiment, compound D is administered once daily. In another embodiment, compound D is administered twice daily. In yet another embodiment, compound D provided herein is administered three times per day. In yet another embodiment, compound D provided herein is administered four times per day. In yet another embodiment, compound D provided herein is administered once every other day. In yet another embodiment, compound D provided herein is administered twice weekly. In yet another embodiment, compound D provided herein is administered once weekly. In yet another embodiment, compound D provided herein is administered every two weeks. In yet another embodiment, compound D provided herein is administered once every three weeks. In yet another embodiment, compound D provided herein is administered once every four weeks.

In certain embodiments, the compound D formulations provided herein are administered once daily from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks. In certain embodiments, the compound D formulations provided herein are applied once daily for one, two, three, or four weeks. In one embodiment, the compound D formulations provided herein are applied once daily for 1 day. In one embodiment, a compound D formulation provided herein is applied once daily for 2 days. In one embodiment, a compound D formulation provided herein is applied once daily for 3 days. In one embodiment, a compound D formulation provided herein is applied once daily for 4 days. In one embodiment, a compound D formulation provided herein is applied once daily for 5 days. In one embodiment, a compound D formulation provided herein is applied once daily for 6 days. In one embodiment, the compound D formulations provided herein are applied once daily for one week. In one embodiment, the compound D formulations provided herein are applied once daily for up to 10 days. In another embodiment, a compound D formulation provided herein is applied once daily for two weeks. In yet another embodiment, a compound D formulation provided herein is applied once daily for three weeks. In yet another embodiment, a compound D formulation provided herein is applied once daily for four weeks.

Combination therapy

In one embodiment, provided herein is a method of treating, preventing and/or managing cancer, comprising administering to a patient compound D in combination with one or more second agents selected from the group consisting of: a JAK inhibitor, a FLT3 inhibitor, an mTOR inhibitor, a spliceosome inhibitor, a BET inhibitor, an SMG1 inhibitor, an ERK inhibitor, a LSD1 inhibitor, a BH3 mimetic, a topoisomerase inhibitor, and a RTK inhibitor, and optionally in combination with radiotherapy, blood transfusion, or surgery. Examples of second active agents are disclosed herein.

In one embodiment, provided herein is a method of treating, preventing and/or managing cancer, comprising administering to a patient a compound D formulation provided herein in combination with one or more second active agents, and optionally in combination with radiation therapy, blood transfusion or surgery. Examples of second active agents are disclosed herein.

As used herein, the term "combination" includes the use of more than one therapy (e.g., one or more prophylactic and/or therapeutic agents). However, use of the term "combination" does not limit the order in which therapies (e.g., prophylactic and/or therapeutic agents) are administered to a patient having a disease or disorder. For example, "combining" may include applying as a mixture, applying simultaneously using separate formulations, and applying sequentially in any order. By "continuous" is meant that a specified time elapses between administration of the active agent. For example, "continuously" may be more than 10 minutes elapsed between administration of the individual active agents. Thus, the time period may be more than 10min, more than 30 minutes, more than 1 hour, more than 3 hours, more than 6 hours, or more than 12 hours. For example, a first therapy (e.g., a prophylactic or therapeutic agent, such as a compound D formulation provided herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy (e.g., a prophylactic or therapeutic agent) to a subject. Triple combination therapy is also contemplated herein.

In one embodiment, administration of compound D (including the compound D formulations provided herein) and one or more second active agents to a patient may be performed simultaneously or sequentially by the same or different routes of administration. In one embodiment, administration of compound D (including the compound D formulations provided herein) and one or more second active agents to a patient may be performed simultaneously or sequentially by the same or different routes of administration. The suitability of a particular route of administration for a particular active agent will depend on the active agent itself (e.g., whether it can be administered orally without breaking down prior to entering the bloodstream) and the cancer being treated.

The route of administration of compound D (including the compound D formulations provided herein) is independent of the route of administration of the second therapy. Thus, in one embodiment, compound D (including the compound D formulations provided herein) is administered intravenously, and the second therapy can be administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraocularly, locally delivered by catheter or stent, subcutaneously, intraadiposally, intraarticularly, intrathecally, or in a sustained release dosage form. In one embodiment, compound D (including the compound D formulations provided herein) and the second therapy are administered by the same mode of administration (by IV). In another embodiment, compound D (including the compound D formulations provided herein) is administered by one mode of administration (e.g., by IV) and the second agent (anti-cancer agent) is administered by another mode of administration (e.g., orally).

In one embodiment, the second active agent is administered intravenously or subcutaneously and in an amount of from about 1 to about 1000mg, from about 5 to about 500mg, from about 10 to about 350mg, or from about 50 to about 200mg once or twice daily. The specific amount of the second active agent will depend on the specific agent used, the type of disease being treated and/or controlled, the severity and stage of the disease, and the amount of compound D and any optional additional active agents concurrently administered to the patient.

One or more second active ingredients or agents may be used with compound D in the methods and compositions provided herein. The second active agent can be a macromolecule (e.g., a protein) or a small molecule (e.g., a synthetic inorganic, organometallic, or organic molecule).

Examples of macromolecular active agents include, but are not limited to, hematopoietic growth factors, cytokines, and monoclonal and polyclonal antibodies, particularly therapeutic antibodies against cancer antigens. Typical macromolecular active agents are biomolecules, such as naturally occurring or synthetic or recombinant proteins. Proteins that are particularly useful in the methods and compositions provided herein include proteins that stimulate the survival and/or proliferation of hematopoietic precursor cells and immunocompetent producing cells in vitro or in vivo. Other useful proteins stimulate the division and differentiation of committed erythroid progenitors in cells in vitro or in vivo. Specific proteins include, but are not limited to: interleukins such as IL-2 (including recombinant IL-II ("rIL 2") and canarypox IL-2), IL-10, IL-12 and IL-18; interferons such as interferon alpha-2 a, interferon alpha-2 b, interferon alpha-n 1, interferon alpha-n 3, interferon beta-Ia, and interferon gamma-Ib; GM-CF and GM-CSF; and EPO.

In certain embodiments, the GM-CSF, G-CSF, SCF, or EPO is present at about 1 to about 750mg/m for about five days in a four or six week cycle ² About 25 to about 500mg/m per day ² About 50 to about 250mg/m per day ² Daily or from about 50 to about 200mg/m ² Amounts in the range of a day were administered subcutaneously. In certain embodiments, GM-CSF may be present at about 60 to about 500mcg/m ² In an amount of from about 5 to about 12mcg/m intravenously over 2 hours ² Subcutaneous administration on day. In certain embodiments, G-CSF can be initially administered subcutaneously in an amount of about 1 mcg/kg/day and can be modulated according to an increase in total granulocyte count. Maintenance doses of G-CSF can be administered subcutaneously in amounts of about 300 (in smaller patients) or 480 mcg. In certain embodiments, EPO can be administered subcutaneously in an amount of 10,000 units per week 3And (4) performing secondary application.

Specific proteins that may be used in the methods and compositions include, but are not limited to: filgrastim, tradename in the united states

(Amgen, Qianzuan, Calif.); sagnathitin, tradename in the United states

(Immunex, seattle, washington); and recombinant EPO, which is available in the United states under the trade name

(Amgen, Qianzuan, Calif.) is sold.

Recombinant and mutant forms of GM-CSF can be prepared, for example, as described in U.S. Pat. Nos. 5,391,485; 5,393,870 and 5,229,496, all of which are incorporated herein by reference. Recombinant and mutant forms of G-CSF can be identified, for example, in U.S. patent nos. 4,810,643; 4,999,291, respectively; 5,528,823 and 5,580,755, which are all incorporated herein by reference in their entirety.

Also provided for use in combination with compound D (including compound D formulations) are natural, naturally occurring, and recombinant proteins. Further contemplated are mutants and derivatives (e.g., modified forms) of naturally occurring proteins that exhibit at least some of the pharmacological activity of the proteins on which they are based in vivo. Examples of mutants include, but are not limited to, proteins having one or more amino acid residues that are different from the corresponding residues in the naturally occurring form of the protein. The term "mutant" also encompasses proteins that lack a carbohydrate moiety that is normally present in its naturally occurring form (e.g., a non-glycosylated form). Examples of derivatives include, but are not limited to, pegylated derivatives and fusion proteins, such as proteins formed by fusing IgG1 or IgG3 to the protein of interest or an active portion of the protein. See, e.g., penechet, m.l. and Morrison, s.l., j.immunol.methods248:91-101 (2001).

Antibodies that can be used in combination with compound D (including the compound D formulations provided herein) include monoclonal antibodies and polyclonal antibodies. Examples of antibodies include, but are not limited to, trastuzumab

Rituximab

Bevacizumab (Avastin) ^TM ) Pertuzumab (Omnitarg) ^TM ) Tositumomab

Epilozumab emluo

And G250. Compound D formulations may also be combined with anti-TNF-alpha antibodies and/or anti-EGFR antibodies (e.g., such as

Or panitumumab) or combinations thereof.

The macromolecular active agent may be administered in the form of an anti-cancer vaccine. For example, vaccines that secrete or cause secretion of cytokines such as IL-2, G-CSF, and GM-CSF can be used in the provided methods and pharmaceutical compositions. See, e.g., Emens, L.A., et al, curr. opinion mol. ther.3(1):77-84 (2001).

The second active agent, which is a small molecule, may also be used to mitigate adverse effects associated with the application of the compound D formulations provided herein. However, as with some macromolecules, many are believed to be capable of providing a synergistic effect when applied together (e.g., before, after, or simultaneously) with compound D (compound D formulations provided herein). Examples of small molecule second active agents include, but are not limited to, anti-cancer agents, antibiotics, immunosuppressive agents, and steroids.

In certain embodiments, the second agent is an HSP inhibitor, a proteasome inhibitor, an FLT3 inhibitor, or an mTOR inhibitor. In some embodiments, the mTOR inhibitor is an mTOR kinase inhibitor.

Examples of anti-cancer agents for use in the methods or compositions described herein include, but are not limited to: acivicin; aclarubicin; (ii) aristozole hydrochloride; (ii) abelmoscine; (ii) Alexanox; aldesleukin; altretamine; an apramycin; amenthraquinone acetate; amsacrine; anastrozole; an anthracycline; an asparaginase enzyme; a triptyline; azacitidine; azatepa; a nitrogenous mycin; batimastat; benzotepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate (bisnafide dimesylate); bizelesin; bleomycin sulfate; brequinar sodium; briprimine; busulfan; actinomycin; (ii) carpoterone; (ii) a karanamide; a carbapenem; carboplatin; carmustine; a doxorubicin hydrochloride; folding to get new; cediogo, and cediogo; celecoxib (COX-2 inhibitor); chlorambucil; a sirolimus; cisplatin; cladribine; clofarabine; cllinaltone mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunomycin hydrochloride; decitabine; (ii) dexomaplatin; tizanoguanine; dizyguanine mesylate; diazaquinone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; drotandrosterone propionate; daptomycin; edatrexae; (ii) nilisil hydrochloride; elsamitrucin; enloplatin; an enpu urethane; epinastine; epirubicin hydrochloride; (ii) ebuzole; isosbacin hydrochloride; estramustine; estramustine sodium phosphate; etanidazole; etoposide; etoposide phosphate; etoposide (etoprine); fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; (iii) flucitabine; a phosphorus quinolone; fostrexasin sodium; gemcitabine; gemcitabine hydrochloride; a hydroxyurea; idarubicin hydrochloride; ifosfamide; ilofovir dipivoxil; iproplatin; irinotecan; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprorelin acetate; liazole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; (ii) maxolone; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; (ii) a melanoril; mercaptopurine; methotrexate; methotrexate sodium; chlorpheniramine; meurtripypde; mitodomide; mitocarcin (mitocarcin); mitorubin (mitocromin); mitogen (mitogillin); mitomacin; mitomycin; mitospirane culturing; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; a noggin; omasitaxine (omaetaxine); ormaplatin; oshuzuren; paclitaxel; a pemetrexed; a calicheamicin; pentazocine; pellomycin sulfate; cultivating phosphoramide; pipobroman; piposulfan; pyrrole anthraquinone hydrochloride; (ii) a plicamycin; pramipexole (plomestane); porfimer sodium; poiseuilrocycin; deltemustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazole furan rhzomorph; (ii) lybodenosine; safrog; safrog hydrochloride; semustine; hyperbolic qin; sorafenib; sodium phosphonoaspartate (sparfosate sodium); a sparamycin; helical germanium hydrochloride; spiromustine; spiroplatinum; streptomycin; streptozotocin; a sulfochlorophenylurea; a talithromycin; sodium tegafur; taxotere; tegafur; tiloxanthraquinone hydrochloride; temoporfin; (ii) teniposide; a tiroxiron; a testosterone ester; (ii) a thiopurine; thioguanine; thiotepa; thiazolfurin (tiazofurin); tirapazamine; toremifene citrate; triton acetate; triciribine phosphate; trimetrexate; tritroximate glucuronate; triptorelin; tobramzole hydrochloride; uramustine; uretipi; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vincristine sulfate; vinorelbine tartrate; vinblastine sulfate; vinzolidine sulfate; (ii) vorozole; zeniplatin; 1, neat setastine; and zorubicin hydrochloride.

Other anti-cancer drugs included in the methods herein include, but are not limited to: 20-epi-1, 25-dihydroxyvitamin D3; 5-acetyleneuropyrimidine; abiraterone; aclacinomycin; acylfulvenes (acylfulvenes); adenosylpentanol; (ii) Alexanox; aldesleukin; ALL-TK antagonist; altretamine; amifostine; (ii) amidox; amifostine; (ii) aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; an angiogenesis inhibitor; an antagonist D; an antagonist G; andrelix (antarelix); anti-dorsal morphogenetic protein-1; anti-androgens, prostate cancer; an antiestrogen; an anti-tumor substance; an antisense oligonucleotide; aphidicolin; an apoptosis gene modulator; a modulator of apoptosis; a purine-free nucleic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestan; amoxicillin; axinatatin 1; axinatatin 2; axinstatin 3; azacinolone; azatoxin; diazotyrosine; baccatin III derivatives; balanol; batimastat; a BCR/ABL antagonist; benzo-dihydroporphin; benzoyl staurosporine; beta lactam derivatives; beta-alethine; betacylomycin B; betulinic acid; a bFGF inhibitor; bicalutamide; a bisantrene group; bis-aziridinyl spermine; a bis-naphthalene method; bisttratene A; bizelesin; brefflate; briprimine; titanium is distributed; buthionine sulfoximine; calcipotriol; calphos protein C; a camptothecin derivative; capecitabine; carboxamide-aminotriazole; carboxamide triazoles; CaRest M3; CARN 700; a cartilage derived inhibitor; folding to get new; casein kinase Inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; (ii) chlorolins; chloroquinoxaline sulfonamide; (ii) cicaprost;

A cis-porphyrin; cladribine; clomiphene analogs; clotrimazole; colismycin A; colismycin B; combretastatin a 4; a combretastatin analog; a concanagen; crambescidin 816; clinatot; nostoc 8; a nostoc a derivative; curve A; cyclopentaquinone; cycloplatam; cephamycin (cypemycin); cytarabine octadecyl phosphate; a cytolytic factor; hexestrol phosphate (cytostatin); daclizumab; decitabine; dehydromembrane ecteinascidin B; dessertraline; dexamethasone; (ii) dexifosfamide; dexrazoxane; (ii) verapamil; diazaquinone; a sphingosine B; didox; diethyl norspermine; dihydro-5-azacytidine; 9-dihydrotaxol; a dioxamycin; diphenylspiromustine; docetaxel; behenyl alcohol; dolasetron; deoxyfluorouridine; doxorubicin; droloxifene; dronabinol; duocarmycin SA; ebselen; etokomustine; edifulin; epidolumab; (ii) nilotinib; elemene; ethirimuron fluoride; epirubicin; epristeride; an estramustine analogue; an estrogen agonist; an estrogen antagonist; etanidazole; etoposide phosphate; exemestane; fadrozole; fa zao Preparing a larabin; fenretinide; filgrastim; finasteride; flavopiridol (flavopiridol); flutemastine (flezelastine); a flashterone; fludarabine; fluxofenacin hydrochloride; fowler; formestane; fostrexed; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; (ii) a gelatinase inhibitor; gemcitabine; a glutathione inhibitor; hepsulfam; heregulin (heregulin); hexamethylene bisamide; hypericin; ibandronic acid; idarubicin; idoxifene; iloperidone; ilofovir dipivoxil; ilomastat; the amount of imatinib (e.g.,

) (ii) a Imiquimod; immunostimulatory peptides; insulin-like growth factor-1 receptor inhibitors; an interferon agonist; an interferon; an interleukin; iodophenylguanidine; iomycin; 4-sweet potato picrol; iprop; 2, according to sorafenib; isobengazole; isohomohalicondrin B; itasetron; jasminoidin lactone (jasplakinolide); kahalalide F; lamellarin triacetate-N; lanreotide; rapamycin (leinamycin); leguminous kiosks; sulfuric acid lentinan; leptin statin; letrozole; leukemia inhibitory factor; leukocyte interferon-alpha; leuprorelin + estrogen + progesterone; leuprorelin; levamisole; liazole; a linear polyamine analog; a lipophilic glycopeptide; a lipophilic platinum compound; lissoclinamide 7; lobaplatin; earthworm phosphatide; lometrexol; lonidamine; losoxanthraquinone; loxoribine; lurtotecan; lutetium texaphyrinate; lisophylline (lysofylline); cleaving the peptide; maytansine; mannostatin a (mannostatin a); marimastat; (ii) maxolone; a silk inhibin; a matriptase inhibitor; a matrix metalloproteinase inhibitor; (ii) a melanoril; mebarone (merbarone); meterelin (meterelin); methioninase; metoclopramide (metoclopramide); an inhibitor of MIF; mifepristone; miltefosine; a Millisetil; mitoguazone; dibromodulcitol; mitomycin analogs; mitonaphthylamine; mitoxin (mitotoxin) fibroblast growth factor-saporin; mitoxantrone; mofagotine; molgramostim (molgramostim); erbitux (Erbitux) and human chorionic gonadotropin A sex hormone; monophosphoryl lipid a + mycobacterial cell wall sk; mopidanol; mustard anticancer agent; mecaprost b (mycaperoxide b); a mycobacterial cell wall extract; myriaporone; n-acetyldinaline; an N-substituted benzamide; nafarelin; nagestip; naloxone + tebuconazole; napavin (napavin); naphterpin; a nartostim; nedaplatin; nemorubicin; neridronic acid; nilutamide; nisamycin; a nitric oxide modulator; a nitrogen oxide antioxidant; nitrulyn; olimoesen cell

O ⁶ -benzylguanine; octreotide; okadsone (okicenone); an oligonucleotide; onapristone; ondansetron; ondansetron; oracin; an oral cytokine inducer; ormaplatin; an oxateclone; oxaliplatin; oxanonomycin; paclitaxel; a paclitaxel analog; a paclitaxel derivative; pamolamine (palaamine); palmitoyl rhizoxin; pamidronic acid; panaxytriol; panomifen; a parabencin; pazelliptin (pazelliptine); a pemetrexed; pedasine (peldesine); sodium pentosan polysulfate; pentostatin; (ii) pentazole; teflon (perfluron); cultivating phosphoramide; perillyl alcohol; phenylazenomycin (phenozinnomycin); a salt of phenylacetic acid; a phosphatase inhibitor; bicibanil (picibanil); pilocarpine hydrochloride; pirarubicin; pirtroxine; placetin A; placetin B; a plasminogen activator inhibitor; a platinum complex; a platinum compound; a platinum-triamine complex; porfimer sodium; poiseuilrocycin; prednisone; propyl bisacridone; prostaglandin J2; a proteasome inhibitor; protein a-based immunomodulators; inhibitors of protein kinase C; protein kinase C inhibitors, microalgae; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurin; pyrazoloacridine; a glycoxylated hemoglobin polyoxyethylene conjugate; a raf antagonist; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; a ras inhibitor; ras-GAP inhibitors; demethylated reteplatin; rhenium (Re) 186 etidronate; rhizomycin; a ribozyme; RII vitamin carboxamide; rohituine (rohitukine); romotede; loquimex; rubiginone B1; rubox yl; safrog; sarin tropin (saintopin); SarCNU; myophyllol a; sargrastim; sdi1 mimetics; semustine; an aging source inhibitor 1; a sense oligonucleotide; a signal transduction inhibitor; sisofilan; sobuzoxane (sobuzoxane); sodium boron carbonate; sodium phenylacetate; solverol; a growth regulatory protein binding protein; sonamin (sonermin); phosphonowinter acid (spartic acid); spicamycin d (spicamycin d); spiromustine; spleen pentapeptide (splenopntin); spongistatin (spongistatin) 1; squalamine; stitiamide (stiiamide); a stromelysin inhibitor; sulfinosine; a superactive vasoactive intestinal peptide antagonist; (ii) surfasta; suramin; swainsonine; tamustine; tamoxifen methyl iodide; taulomustine; tazarotene; sodium tegafur; tegafur; telluropyrylium; a telomerase inhibitor; temoporfin; (ii) teniposide; tetrachlorodecaoxide; tetrazomine; (ii) a thioablistatin; thiocoraline (thiocoraline); thrombopoietin; thrombopoietin mimetics; thymalfasin (Thymalfasin); a thymopoietin receptor agonist; thymotreonam; thyroid stimulating hormone; tin ethyl protoporphyrin (tin ethyl ethylpururin); tirapazamine; cyclopentadienyl titanium dichloride; topstein; toremifene; a translation inhibitor; tretinoin; triacetyl uridine; (iii) triciribine; trimetrexate; triptorelin; tropisetron; toleromide; tyrosine kinase inhibitors; a tyrosine phosphorylation inhibitor; an UBC inhibitor; ubenimex; urogenital sinus derived growth inhibitory factor; a urokinase receptor antagonist; vapreotide; variolin B; vilareol; veratramine; verdins; verteporfin; vinorelbine; vinfosine (vinxaline); vitaxin; (ii) vorozole; zanoteron; zeniplatin; benzalvitamin c (zilascorb); and neat stastatin ester.

In certain embodiments, the second agent is selected from one or more checkpoint inhibitors. In one embodiment, in the methods provided herein, a checkpoint inhibitor is used in combination with compound D or compound D formulations. In another embodiment, in combination with the methods provided herein, two checkpoint inhibitors are used in combination with compound D or a compound D formulation. In yet another embodiment, three or more checkpoint inhibitors are used in combination with compound D or compound D formulations in conjunction with the methods provided herein.

As used herein, the term "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that reduces, inhibits, interferes with, or modulates, in whole or in part, one or more checkpoint proteins. Without being bound by a particular theory, the checkpoint protein modulates T cell activation or function. A number of checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD 86; and PD-1 with its ligands PD-Ll and PD-L2(Pardol, Nature Reviews Cancer,2012,12, 252-264). These proteins appear to be responsible for either costimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins appear to regulate and maintain self-tolerance as well as the duration and magnitude of the physiological immune response. Immune checkpoint inhibitors include antibodies or are derived from antibodies.

In one embodiment, the checkpoint inhibitor is a CTLA-4 inhibitor. In one embodiment, the CTLA-4 inhibitor is an anti-CTLA-4 antibody. Examples of anti-CTLA-4 antibodies include, but are not limited to, U.S. patent nos.: 5,811,097, respectively; 5,811,097; 5,855,887, respectively; 6,051,227, respectively; 6,207,157, respectively; 6,682,736; 6,984,720 and 7,605,238, which are all incorporated herein in their entirety. In one embodiment, the anti-CTLA-4 antibody is tremelimumab (also known as ticilimumab or CP-675,206). In another embodiment, the anti-CTLA-4 antibody is Yipimema (also known as MDX-010 or MDX-101). The pleoman is a fully human monoclonal IgG antibody that binds to CTLA-4. Easily priomam under the trade name Yervoy ^TM And (5) selling.

In one embodiment, the checkpoint inhibitor is a PD-1/PD-L1 inhibitor. Examples of PD-L/PD-L1 inhibitors include, but are not limited to, those described in U.S. patent nos. 7,488,802; 7,943,743, respectively; 8,008,449; 8,168,757, respectively; 8,217,149 and PCT patent application publication nos. WO 2003042402, WO 2008156712, WO 2010089411, WO 2010036959, WO 2011066342, WO 2011159877, WO 2011082400 and WO 2011161699, all of which are incorporated herein in their entirety.

In one embodiment, the checkpoint inhibitor is a PD-1 inhibitor. In a fruitIn embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In one embodiment, the anti-PD-1 antibody is BGB-A317, nivolumab (also known as ONO-4538, BMS-936558, or MDX1106), or pembrolizumab (also known as MK-3475, SCH 900475, or lambrolizumab). In one embodiment, the anti-PD-1 antibody is nivolumab. Navolumab is a human IgG4 anti-PD-1 monoclonal antibody and is sold under the name Opdivo ^TM And (5) selling. In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 antibody and is sold under the tradename Keytruda ^TM And (5) selling. In yet another embodiment, the anti-PD-1 antibody is the humanized antibody CT-011. CT-011 administered alone failed to show a response in treating Acute Myeloid Leukemia (AML) at relapse. In yet another embodiment, the anti-PD-1 antibody is the fusion protein AMP-224. In another embodiment, the PD-1 antibody is BGB-a 317. BGB-a317 is a monoclonal antibody in which the ability to bind Fc γ receptor I is specifically engineered, and which has unique binding characteristics to PD-1, high affinity and superior target specificity.

In one embodiment, the checkpoint inhibitor is a PD-L1 inhibitor. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody. In one embodiment, the anti-PD-L1 antibody is MEDI4736 (devolizumab). In another embodiment, the anti-PD-L1 antibody is BMS-936559 (also known as MDX-1105-01). In yet another embodiment, the PD-L1 inhibitor is atelizumab (also known as MPDL3280A and

)。

in one embodiment, the checkpoint inhibitor is a PD-L2 inhibitor. In one embodiment, the PD-L2 inhibitor is an anti-PD-L2 antibody. In one embodiment, the anti-PD-L2 antibody is rHIgM12B 7A.

In one embodiment, the checkpoint inhibitor is a lymphocyte activation gene-3 (LAG-3) inhibitor. In one embodiment, the LAG-3 inhibitor is soluble Ig fusion protein IMP321(Brignone et al, J.Immunol.,2007,179, 4202-one 4211). In another embodiment, the LAG-3 inhibitor is BMS-986016.

In one embodiment, the checkpoint inhibitor is a B7 inhibitor. In one embodiment, the B7 inhibitor is a B7-H3 inhibitor or a B7-H4 inhibitor. In one embodiment, the B7-H3 inhibitor is the anti-B7-H3 antibody MGA271(Loo et al, clin cancer res.,2012,3834).

In one embodiment, the checkpoint inhibitor is a TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitor (Fourcade et al, j.exp.med.,2010,207,2175-86; Sakuishi et al, j.exp.med.,2010,207,2187-94).

In one embodiment, the checkpoint inhibitor is an OX40(CD134) agonist. In one embodiment, the checkpoint inhibitor is an anti-OX 40 antibody. In one embodiment, the anti-OX 40 antibody is anti-OX-40. In another embodiment, the anti-OX 40 antibody is MEDI 6469.

In one embodiment, the checkpoint inhibitor is a GITR agonist. In one embodiment, the checkpoint inhibitor is an anti-GITR antibody. In one embodiment, the anti-GITR antibody is TRX 518.

In one embodiment, the checkpoint inhibitor is a CD137 agonist. In one embodiment, the checkpoint inhibitor is an anti-CD 137 antibody. In one embodiment, the anti-CD 137 antibody is urilizumab. In another embodiment, the anti-CD 137 antibody is PF-05082566.

In one embodiment, the checkpoint inhibitor is a CD40 agonist. In one embodiment, the checkpoint inhibitor is an anti-CD 40 antibody. In one embodiment, the anti-CD 40 antibody is CF-870,893.

In one embodiment, the checkpoint inhibitor is recombinant human interleukin-15 (rhIL-15).

In one embodiment, the checkpoint inhibitor is an IDO inhibitor. In one embodiment, the IDO inhibitor is INCB 024360. In another embodiment, the IDO inhibitor is indoximod.

In certain embodiments, the combination therapies provided herein comprise two or more checkpoint inhibitors (including checkpoint inhibitors of the same or different classes) as described herein. In addition, the combination therapies described herein can be used in combination with a second active agent as described herein, where appropriate, to treat diseases described herein and understood in the art.

In certain embodiments, compound D can be used in combination with one or more immune cells (e.g., modified immune cells) that express one or more Chimeric Antigen Receptors (CARs) on their surface. Typically, the CAR comprises an extracellular domain from a first protein (e.g., an antigen binding protein), a transmembrane domain, and an intracellular signaling domain. In certain embodiments, once the extracellular domain binds to a target protein, such as a tumor-associated antigen (TAA) or tumor-specific antigen (TSA), a signal is generated by activating the intracellular signaling domain of the immune cell, e.g., to target and kill cells expressing the target protein.

Extracellular domain: the extracellular domain of the CAR binds to an antigen of interest. In certain embodiments, the extracellular domain of the CAR comprises a receptor, or a portion of a receptor, that binds to the antigen. In certain embodiments, the extracellular domain comprises or is an antibody or antigen-binding portion thereof. In particular embodiments, the extracellular domain comprises or is a single chain fv (scfv) domain. The single chain Fv domain may comprise, for example, a VL linked to a VH via a flexible linker, wherein the VL and VH are from an antibody that binds the antigen.

In certain embodiments, the antigen recognized by the extracellular domain of a polypeptide described herein is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). In various embodiments, the tumor-associated antigen or tumor-specific antigen is, but is not limited to, Her2, Prostate Stem Cell Antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, B Cell Maturation Antigen (BCMA), epithelial membrane protein (EMA), Epithelial Tumor Antigen (ETA), tyrosinase, melanoma-24 associated antigen (MAGE), CD19, CD22, CD27, CD30, CD34, CD45, CD70, CD99, CD117, EGFRvIII (epidermal growth factor variant III), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternative reading frame protein), Trp-p8, STEAPI (prostate six transmembrane epithelial antigen 1), chromogranin, cytokeratin, intertyosin, Glial Fibrillary Acidic Protein (GFAP), and alpha-fetoprotein, Macrocystic disease liquid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by lymphocytes; MART-I), myo-D1, Muscle Specific Actin (MSA), neurofilament, Neuron Specific Enolase (NSE), placental alkaline phosphatase, synaptophysin (synaptophysis), thyroglobulin, thyroid transcription factor-1, dimeric form of pyruvate kinase isozyme M2 (tumor M2-PK), abnormal ras protein, or abnormal p53 protein. In certain other embodiments, the TAA or TSA recognized by the extracellular domain of the CAR is integrin α ν β 3(CD61), galactin, or Ral-B.

In certain embodiments, the TAA or TSA recognized by the extracellular domain of the CAR is a cancer/testis (CT) antigen, e.g., BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ES0-1, NY-SAR-35, OY-TES-1, SPANXBI, SPA17, SSX, SYCPI, or TPTE.

In certain other embodiments, the TAA or TSA recognized by the extracellular domain of the CAR is a carbohydrate or ganglioside, e.g., fuc-GMI, GM2 (cancer embryonic antigen-immunogen-1; OFA-I-1); GD2(OFA-I-2), GM3, GD3, etc.

In certain other embodiments, the TAA or TSA recognized by the extracellular domain of the CAR is alpha-actinin-4, Bage-l, BCR-ABL, Bcr-ABL fusion protein, beta-catenin, CA 125, CA 15-3(CA27.29\ BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-l, dek-can fusion protein, EBNA, EF2, Epstein-Barr virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA-All, hsp70-2, KIAA0205, Mart2, Mum-1, 2 and 3, neo-PAP, myosin class I, RAR-9, Ga pml-alpha fusion protein, PRK, K-ras, triose 4, phosphoisomerase,

PTras

4, 6, 7. GnTV, Herv-K-Mel, Lage-1, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, TRP2-Int2, gp100(Pmel17), tyrosinase, TRP-1, TRP-2, MAGE-l, MAGE-3, RAGE, GAGE-l, GAGE-2, p15(58), RAGE, SCP-1, Hom/Mel-40, PRAME, p53, HRas, HER-2/neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Human Papilloma Virus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p 185B 2, p 180B-3, c-met, PSA-1 nm, PSA-19-72, PSA-72-CA, TAB-72, TAB-72, and TAB-4, CAM 17.1, NuMa, K-ras, 13-catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\ KP1, C0-029, FGF-5, G250, Ga733(EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB 70K, NY-C0-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAAG 72, TLP or TPS.

In various embodiments, the tumor-associated antigen or tumor-specific antigen is an AML-associated tumor antigen, as described in s.anguille et al, leukamia (2012),26, 2186-.

Other tumor-associated and tumor-specific antigens are known to those skilled in the art.

TSA and TAA binding receptors, antibodies and scfvs useful for the construction of chimeric antigen receptors are known in the art, as are the nucleotide sequences encoding them.

In certain embodiments, the antigen recognized by the extracellular domain of the chimeric antigen receptor is an antigen that is not normally considered a TSA or TAA but is still associated with tumor cells or damage caused by a tumor. In certain embodiments, for example, the antigen is, e.g., a growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis. Such growth factors, cytokines or interleukins may include, for example, Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), Hepatocyte Growth Factor (HGF), insulin-like growth factor (IGF), or interleukin 8 (IL-8). Tumors can also produce a hypoxic environment locally at the tumor. Thus, in other embodiments, the antigen is a hypoxia-associated factor, such as HIF-1 α, HIF-1 β, HIF-2 α, HIF-2 β, HIF-3 α, or HIF-3 β. Tumors can also cause local damage to normal tissues, resulting in the release of molecules known as damage-associated molecular pattern molecules (DAMPs; also known as sirens). Thus, in certain other embodiments, the antigen is a DAMP, such as heat shock protein, chromatin-associated protein high mobility group box 1(HMGB 1), S100A8(MRP8, calgranulin a), S100a9(MRP14, calgranulin B), serum amyloid a (saa), or may be deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.

Transmembrane domain: in certain embodiments, the extracellular domain of the CAR is linked to the transmembrane domain of the polypeptide by a linker, spacer, or hinge polypeptide sequence (e.g., a sequence from CD28 or a sequence from CTLA 4). The transmembrane domain may be obtained or derived from the transmembrane domain of any transmembrane protein, and may comprise all or part of such a transmembrane domain. In particular embodiments, the transmembrane domain may be obtained or derived from, for example, CD8, CD16, cytokine receptors, and interleukin receptors or growth factor receptors, among others.

Intracellular signaling domain: in certain embodiments, the intracellular domain of the CAR is or comprises an intracellular domain or motif of a protein that is expressed on the surface of a T cell and triggers activation and/or proliferation of the T cell. Such domains or motifs are capable of transmitting a primary antigen binding signal necessary for activating T lymphocytes in response to binding of an antigen to the extracellular portion of the CAR. Typically, this domain or motif comprises or is ITAM (immunoreceptor tyrosine-based activation motif). ITAM-containing polypeptides suitable for use in a CAR include, for example, the zeta CD3 chain (CD3 zeta) or an ITAM-containing portion thereof. In a specific embodiment, the intracellular domain is a CD3 ζ intracellular signaling domain. In other embodiments, the intracellular domain is from a lymphocyte receptor chain, a TCR/CD3 complex protein, a Fe receptor subunit, or an IL-2 receptor subunit. In certain embodiments, the CAR further comprises one or more co-stimulatory domains or motifs, e.g., as part of the intracellular domain of the polypeptide. The one or more co-stimulatory domains or motifs may be or may comprise one or more of: a co-stimulatory CD27 polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, a co-stimulatory OX40(CD134) polypeptide sequence, a stimulatory 4-1BB (CD137) polypeptide sequence, or a co-stimulatory inducible T cell co-stimulatory (ICOS) polypeptide sequence, or other co-stimulatory domains or motifs, or any combination thereof.

The CAR may further comprise a T cell survival motif. The T cell survival motif can be any polypeptide sequence or motif that promotes T lymphocyte survival upon stimulation by an antigen. In certain embodiments, the T cell survival motif is or is derived from CD3, CD28, an intracellular signaling domain of an IL-7 receptor (IL-7R), an intracellular signaling domain of an IL-12 receptor, an intracellular signaling domain of an IL-15 receptor, an intracellular signaling domain of an IL-21 receptor, or an intracellular signaling domain of a transforming growth factor beta (TGF β) receptor.

The modified immune cell expressing the CAR can be, for example, a T lymphocyte (a T cell, e.g., a CD4+ T cell or a CD8+ T cell), a cytotoxic lymphocyte (CTL), or a Natural Killer (NK) cell. The T lymphocytes used in the compositions and methods provided herein can be naive T lymphocytes or MHC-restricted T lymphocytes. In certain embodiments, the T lymphocyte is a Tumor Infiltrating Lymphocyte (TIL). In certain embodiments, the T lymphocytes have been isolated from a tumor biopsy, or have been expanded from T lymphocytes isolated from a tumor biopsy. In certain other embodiments, the T cells have been expanded by T lymphocytes isolated from or from peripheral blood, cord blood, or lymph fluid. The immune cells to be used to generate modified immune cells expressing the CAR can be isolated using conventional methods recognized in the art, such as blood collection followed by apheresis and optional antibody-mediated cell separation or sorting.

The modified immune cells are preferably autologous to the individual to whom the modified immune cells are to be administered. In certain other embodiments, the modified immune cells are allogeneic to the individual to whom the modified immune cells are to be administered. In using allogeneic T lymphocytes or NK cells to prepare modified T lymphocytes, it is preferred to select T lymphocytes or NK cells that will reduce the likelihood of an individual developing Graft Versus Host Disease (GVHD). For example, in certain embodiments, virus-specific T lymphocytes are selected for use in making modified T lymphocytes; such lymphocytes would be expected to bind to any receptor antigen and thus would have greatly reduced natural ability to be activated by it. In certain embodiments, receptor-mediated rejection of allogeneic T lymphocytes may be reduced by co-administering to the host one or more immunosuppressive agents (e.g., cyclosporine, tacrolimus, sirolimus, cyclophosphamide, etc.).

T lymphocytes (e.g., unmodified T lymphocytes or T lymphocytes expressing CD3 and CD28 or comprising a polypeptide comprising a CD3 zeta signaling domain and a CD28 costimulatory domain) can be expanded using antibodies against CD3 and CD28 (e.g., antibodies attached to beads); see, for example, U.S. patent nos. 5,948,893; 6,534,055, respectively; 6,352,694, respectively; 6,692,964, respectively; 6,887,466, and 6,905,681.

The modified immune cells (e.g., modified T lymphocytes) can optionally comprise a "suicide gene" or "safety switch" that is capable of killing substantially all of the modified immune cells when desired. For example, in certain embodiments, the modified T lymphocyte can comprise an HSV thymidine kinase gene (HSV-TK) which, upon contact with ganciclovir, causes the modified T lymphocyte to die. In another embodiment, the modified T lymphocyte comprises an inducible caspase, such as inducible caspase 9(icaspase9), e.g., a fusion protein between caspase9 and human FK506 binding protein, allowing dimerization with specific small molecule drugs. See Straathof et al, Blood 105(11):4247-4254 (2005).

Specific second active agents that may be used in the methods or compositions include, but are not limited to, rituximab, olimerson

Pseudogram (remicade), docetaxel, celecoxib, melphalan and dexamethasone

Steroids, gemcitabine, cisplatin, temozolomide, etoposide, cyclophosphamide, temoda (temodar), carboplatin, procarbazine, gliadel, tamoxifen, topotecan, methotrexate, doxorubicin, and a pharmaceutically acceptable salt thereof,

Taxol (Taxol), taxotere, fluorouracil, leucovorin, irinotecan, receptacle (xelodA), interferon alphA, pegylated interferon alphA (e.g., PEG INTRON-A), capecitabine, cisplatin, thiotepA, fludarabine, carboplatin, liposomal daunomycin, cytarabine, docetaxel, paclitaxel, vinblastine, IL-2, GM-CSF, dacarbazine, vinorelbine, zoledronic acid, pamidronate, clarithromycin (biaxin), busulfan, prednisone, bisphosphonate, arsenic trioxide, vincristine, doxorubicin, leucovorin, and the like

Paclitaxel, ganciclovir, Adriamycin, estramustine sodium phosphate

Sulindac and etoposide.

In certain embodiments of the methods provided herein, the use of a second active agent in combination with compound D (including the compound D formulations provided herein) may be altered or delayed during or shortly after the administration of compound D (including the compound D formulations provided herein), as deemed appropriate by the skilled practitioner. In certain embodiments, subjects administered compound D (including the compound D formulations provided herein), alone or in combination with other therapies, may receive supportive care, including antiemetic, bone marrow growth factor, and platelet transfusions, as appropriate. In some embodiments, a growth factor as a second active agent can be administered to a subject administered compound D (including the compound D formulations provided herein), according to the judgment of a practitioner of skill in the art. In some embodiments, administration of compound D (including compound D formulations provided herein) in combination with erythropoietin or dabbepotein (Aranesp) is provided.

In one aspect, provided herein is a method of treating, preventing, managing and/or ameliorating locally advanced or metastatic transitional cell bladder cancer, the method comprising administering a compound D formulation with gemcitabine, cisplatin, 5-fluorouracil, mitomycin, methotrexate, vinblastine, doxorubicin, carboplatin, thiotepa, paclitaxel, docetaxel, alemtuzumab, avizumab, dewarpuzumab, keytruruda (pembrolizumab) and/or nivolumab.

In one aspect, the methods of treating, preventing, managing, and/or ameliorating cancer provided herein comprise administering a compound D formulation in combination with a second active ingredient that: temozolomide for use in pediatric patients with recurrent or progressive brain tumors or recurrent neuroblastoma; celecoxib, etoposide and cyclophosphamide are used for recurrent or progressive central nervous system cancer; temodar for patients with recurrent or progressive meningiomas, malignant meningiomas, hemangiothecoma, multiple brain metastases, recurrent brain tumors, or newly diagnosed glioblastoma multiforme; irinotecan for use in patients with relapsed glioblastoma; carboplatin for pediatric patients with brainstem glioma; procarbazine for use in pediatric patients with progressive glioblastoma; cyclophosphamide is used in patients with poor prognosis malignant brain tumors, newly diagnosed or recurrent glioblastoma multiforme;

for high-grade recurrent glioblastoma; temozolomide and tamoxifen for anaplastic astrocytomas; or topotecan for glioma, glioblastoma, anaplastic astrocytoma or anaplastic oligodendroglioma.

In one aspect, the methods provided herein for treating, preventing, managing and/or ameliorating metastatic breast cancer comprise administering a compound D formulation with methotrexate, cyclophosphamide, capecitabine, 5-fluorouracil, taxane, temsirolimus, a pharmaceutically acceptable salt, a pharmaceutically acceptable acid, a pharmaceutically acceptable salt, a pharmaceutically acceptable acid, a pharmaceutically acceptable salt, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof,

(paclitaxel protein-binding particles for injection suspension) (albumin-binding), lapatinib, herceptin, disodium pamidronate, eribulin mesylate, everolimus, gexicin(ii) capecitabine, palbociclib, ixabepilone, herculela, pertuzumab, anastrozole, docetaxel, doxorubicin hydrochloride, epirubicin hydrochloride, toremifene, fulvestrant, goserelin acetate, regoracil, megestrol acetate, vinblastine, aromatase inhibitors such as letrozole, exemestane, selective estrogen modulators, estrogen receptor antagonists, anthracyclines, emtansine, and/or pexidantinib.

In one aspect, the methods of treating, preventing, managing and/or ameliorating a neuroendocrine tumor provided herein comprise administering a compound D formulation with at least one of everolimus, avizumab, sunitinib, polygerimer (nexavar), leucovorin, oxaliplatin, temozolomide, capecitabine, bevacizumab, doxorubicin (adriamycin), fluorouracil (Adrucil, 5-fluorouracil), streptozotocin (Zanosar), dacarbazine, tannin (sandostatin), lanreotide and/or pasireotide to a patient having a neuroendocrine tumor.

In one aspect, the methods of treating, preventing, managing, and/or ameliorating metastatic breast cancer provided herein comprise administering a compound D formulation with methotrexate, gemcitabine, cisplatin, cetuximab, 5-fluorouracil, bleomycin, docetaxel, carboplatin, hydroxyurea, pembrolizumab, and/or nivolumab to a patient with recurrent or metastatic head and neck cancer.

In one aspect, the methods of treating, preventing, managing and/or ameliorating pancreatic cancer provided herein comprise administering to a patient having pancreatic cancer a compound D formulation with gemcitabine,

5-fluorouracil, everolimus (afinitor), irinotecan, mitomycin C, sunitinib malate and/or Tarceva (tarceva).

In one aspect, the methods of treating, preventing, managing and/or ameliorating colon or rectal cancer provided herein comprise administering a compound D formulation with

Avastin (avastatin), oxaliplatin, 5-fluorouracil, irinotecan, capecitabine, cetuximab, ramucirumab, panitumumab, bevacizumab, leucovorin calcium, lonsurf, regorafenib, ziv-aflibercept, taxol and/or taxotere.

In one aspect, the methods of treating, preventing, managing, and/or ameliorating refractory colorectal cancer provided herein comprise administering compound D formulation with capecitabine and/or vemurafenib to a patient with refractory colorectal cancer or a patient with failure of first line therapy or underperforming colon or rectal adenocarcinoma.

In one aspect, the methods of treating, preventing, managing and/or ameliorating colorectal cancer provided herein comprise administering a compound D formulation with fluorouracil, leucovorin and/or irinotecan to patients with colorectal cancer (including stages 3 and 4) or patients who have previously been treated for metastatic colorectal cancer therapy.

In certain embodiments, compound D formulations provided herein are administered to a patient having refractory colorectal cancer in combination with capecitabine, hiloda, and/or irinotecan.

In certain embodiments, compound D formulations provided herein are administered to patients with refractory colorectal cancer or patients with unresectable or metastatic colorectal cancer in combination with capecitabine and irinotecan.

In one aspect, the methods provided herein comprise administering to a patient with unresectable or metastatic hepatocellular carcinoma compound D formulation with interferon alpha or capecitabine; or administering compound D formulation with cisplatin and thiotepa or with sorafenib tosylate to a patient with primary or metastatic liver cancer.

In one aspect, the methods provided herein comprise administering a compound D formulation with doxorubicin, paclitaxel, vinblastine, pegylated interferon alfa, and/or recombinant interferon alfa-2 b to a patient with kaposi's sarcoma.

In one aspect, the methods provided herein comprise administering compound D formulation with at least one of enidipine, arsenic trioxide, fludarabine, carboplatin, daunorubicin, cyclophosphamide, cytarabine, doxorubicin, idarubicin, mitoxantrone hydrochloride, thioguanine, vincristine, midostaurin (midostaurin), and/or topotecan to a patient having acute myeloid leukemia, including relapsed or refractory or high risk acute myeloid leukemia.

In one aspect, the methods provided herein comprise administering a compound D formulation with at least one of enzipine, liposomal daunorubicin, topotecan, and/or cytarabine to a patient having an adverse karyotic acute myeloblastic leukemia.

In one aspect, the methods provided herein comprise administering compound D with an IDH2 inhibitor to a patient having leukemia, wherein the leukemia is characterized by the presence of a mutant allele of IDH 2. Exemplary IDH2 inhibitors are disclosed in U.S. patent nos. 9,732,062; 9,724,350, respectively; 9,738,625 and 9,579,324, and U.S. Pat. Nos. 2016-0159771 and 2016-0158230A 1. In one aspect, the methods provided herein comprise administering compound D with enzidipine to a patient having a leukemia, wherein the leukemia is characterized by the presence of a mutant allele of IDH 2. In certain embodiments, the combination of compound D and an IDH2 inhibitor increases differentiated cells (CD34-/CD38) and erythroblasts in a patient with acute myeloid leukemia, wherein the acute myeloid leukemia is characterized by the presence of IDH 2R 140Q. In certain embodiments, the combination of compound D and an IDH2 inhibitor reduces progenitor cells (CD34+/CD38+) and HSCs in patients with acute myeloid leukemia, wherein the acute myeloid leukemia is characterized by the presence of IDH 2R 140Q.

In one aspect, the methods provided herein comprise administering compound D with enzidipine to a patient having acute myeloid leukemia, wherein the acute myeloid leukemia is characterized by the presence of a mutant allele of IDH 2. In one embodiment, the mutant allele of IDH2 is IDH 2R 140Q or R172K.

In one aspect, the methods provided herein comprise administering a compound D formulation with enzidipine to a patient having a leukemia, wherein the leukemia is characterized by the presence of a mutant allele of IDH 2. In one aspect, the methods provided herein comprise administering compound D formulation with enzidipine to a patient having acute myeloid leukemia, wherein the acute myeloid leukemia is characterized by the presence of a mutant allele of IDH 2. In one embodiment, the mutant allele of IDH2 is IDH 2R 140Q or R172K.

In one aspect, the methods provided herein comprise administering compound D with 6- (6- (trifluoromethyl) pyridin-2-yl) -N2- (2- (trifluoromethyl) pyridin-4-yl) -1,3, 5-triazine-2, 4-diamine (compound 2) to a patient having a leukemia, wherein the leukemia is characterized by the presence of a mutant allele of IDH 2. In one aspect, the methods provided herein comprise administering compound D with compound 2 to a patient having acute myeloid leukemia, wherein the acute myeloid leukemia is characterized by the presence of a mutant allele of IDH 2. In one embodiment, the mutant allele of IDH2 is IDH 2R 140Q or R172K.

In one aspect, the methods provided herein comprise administering compound D formulation with compound 2 to a patient having a leukemia, wherein the leukemia is characterized by the presence of a mutant allele of IDH 2. In one aspect, the methods provided herein comprise administering compound D formulation with compound 2 to a patient having acute myeloid leukemia, wherein the acute myeloid leukemia is characterized by the presence of a mutant allele of IDH 2. In one embodiment, the mutant allele of IDH2 is IDH 2R 140Q or R172K.

In one aspect, the methods provided herein comprise administering to a patient having non-small cell lung cancer a compound D formulation with methotrexate, mechlorethamine hydrochloride, afatinib dimaleate, pemetrexed, bevacizumab, carboplatin, cisplatin, ceritinib (ceritinib), crizotinib (crizotinib), ramucirumab, pembrolizumab, docetaxel, vinorelbine tartrate, gemcitabine, meclizine, flutriafol, fluazinam, and combinations thereof,

Erlotinib, gefitinib, irinotecan, everolimus, erlotinib (alectinib), bugatinib, nivolumab, oxitinib (osimertinib), axitinibTezumab ozolomide, rituximab (necitumumab) and/or.

In one aspect, the methods provided herein comprise administering compound D formulation with carboplatin and irinotecan to a patient having non-small cell lung cancer.

In one aspect, the methods provided herein comprise administering a compound D formulation with docetaxel to a patient having non-small cell lung cancer who has been previously treated with carbo/etoposide and radiation therapy.

In one aspect, the methods provided herein comprise administering to a patient having non-small cell lung cancer a combination of compound D formulation with carboplatin and/or taxotere or with carboplatin, paclitaxel, and/or thoracic vertebral radiotherapy.

In one aspect, the methods provided herein comprise administering compound D formulation with taxotere to a patient having stage IIIB or IV non-small cell lung cancer.

In one aspect, the methods provided herein comprise administering compound D formulation with olmoeson to a patient with small cell lung cancer

Methotrexate, mechlorethamine hydrochloride, etoposide, topotecan and/or doxorubicin.

In one aspect, the methods provided herein comprise administering a compound D formulation with venetocix (venetolax), ABT-737(Abbott Laboratories), and/or obacara (obaaclax) (GX15-070) to patients with lymphoma and other blood cancers.

In one aspect, the methods provided herein comprise administering a compound D formulation with a second active ingredient such as vinblastine or fludarabine addictris, ambochlorin, becenum, bleomycin, bevacizumab, carmustine chlorambucil, cyclophosphamide, dacarbazine, doxorubicin, lomustine, procarbazine (mulane), mechlorethamine hydrochloride, prednisone, procarbazine hydrochloride, vincristine, methotrexate, nelarabine (nellabin), belinostat (belinostat), bendamustine HCl, tositumomab and iodo131 tositumomab, interleukin, dexamethasone, prasugrel, tolazamide, tollizumab (orubin), tollizumab (obilizumab), tolazamide (aldicab), tolazamide (beralomycin), dexrazine (berrubicin), tolazamide (berrubicin), tolazalomavine (aldicab), tolazamide (aldicarb), tolazalomavine (aldicab), or a, Ibritumomab tiuxefan (ibritumomab), tiuxefan, ibrutinib (ibritinib), idelarisib (idelasib), interferons (intron a), romidepsin (romidepsin), lenalidomide, rituximab and/or vorinostat.

In one aspect, the methods provided herein comprise administering to a patient having various types or stages of melanoma a compound D formulation with taxotere, dabrafenib, imlygic, caprilomar, pembrolizumab, nivolumab, tremelimumab, vemurafenib, taliomogene lahermaphvec, IL-2, IFN, GM-CSF and/or dacarbazine, aldesleukin, cobicistinib, Intron

Polyethylene glycol interferon alpha-2 b and/or trametinib.

In one aspect, the methods provided herein comprise administering compound D formulation with vinorelbine or disodium pemetrexed to a patient with malignant mesothelioma, or stage IIIB non-small cell lung cancer with pleural graft, or malignant pleural effusion mesothelioma syndrome.

In one aspect, the methods provided herein for treating A patient with various types or stages of multiple myelomA comprise administering A compound D formulation with dexamethasone, zoledronic acid, pamidronate, GM-CSF, clarithromycin, vinblastine, melphalan, busulfan, cyclophosphamide, IFN, prednisone, A bisphosphonate, celecoxib, arsenic trioxide, PEG INTRON-A, vincristine, becenum, bortezomib (bortezomib), carfilzomib, doxorubicin, panobinostat (panobinostat), lenalidomide, pomalidomide (pomlidomide), thalidomide, plerixamide (mozobil), carmustine, dalamumab, elotuzumab (elotuzumab), salzoxamide citrate, plerixafort (plerixafor), or A combination thereof.

In certain embodiments, compound D formulations provided herein are administered to patients with various types or stages of multiple myeloma in combination with Chimeric Antigen Receptor (CAR) T cells. In certain embodiments, the CAR T cells in combination target B Cell Maturation Antigen (BCMA), and in more particular embodiments, the CAR T cells are bb2121 or bb 21217. In some embodiments, the CAR T cell is JCARH 125.

In certain embodiments, provided herein are compound D formulations with doxorubicin

Vincristine and/or dexamethasone

The combination is administered to a patient with relapsed or refractory multiple myeloma.

In certain embodiments, the methods provided herein comprise administering a combination of compound D formulation with taxol, carboplatin, doxorubicin, gemcitabine, cisplatin, hiloda, paclitaxel, dexamethasone, avastin (avastin), cyclophosphamide, topotecan, olaparib (olaparib), thiotepa, melphalan, nilapamide tosylate monohydrate, rukappab (rubraca), or a combination thereof, to a patient with various types or stages of ovarian cancer, such as peritoneal cancer, papillary serous cancer, refractory ovarian cancer, or recurrent ovarian cancer.

In certain embodiments, the methods provided herein comprise administering to a patient having various types or stages of prostate cancer a combination of a compound D formulation with hiloda, 5FU/LV, gemcitabine, irinotecan + gemcitabine, cyclophosphamide, vincristine, dexamethasone, GM-CSF, celecoxib, taxotere, ganciclovir, paclitaxel, doxorubicin, docetaxel, estramustine, Emcyt, denderon, abira (zytiga), bicalutamide, cabazitaxel (cabazitaxel), degarelix (degarelix), enzalutamide (enzalutamide), norrex (zoladex), leuprolide acetate, mitoxantrone hydrochloride, prednisone, sipuleucel-T, radium dichloride 223, or a combination thereof.

In certain embodimentsThe methods provided herein comprise administering to a patient having various types or stages of renal cell carcinoma a compound D formulation with capecitabine, IFN, tamoxifen, IL-2, GM-CSF,

flutamide (flutamide), goserelin acetate, nilutamide, or combinations thereof.

In certain embodiments, the methods provided herein comprise administering a compound D formulation to patients with various types or stages of gynecological, uterine or soft tissue sarcoma cancers in combination with IFN, dactinomycin, doxorubicin, imatinib mesylate, pazopanib hydrochloride, trabectedin, eribulin mesylate, olaratumab, COX-2 inhibitors such as celecoxib, and/or sulindac.

In one aspect, the methods provided herein comprise administering to a patient having a solid tumor of various types or stages a compound D formulation in combination with celecoxib, etoposide, cyclophosphamide, docetaxel, apectibine, IFN, tamoxifen, IL-2, GM-CSF, or a combination thereof.

In one aspect, the methods provided herein comprise administering a compound D formulation in combination with Celebrex (Celebrex), etoposide, cyclophosphamide, docetaxel, apectibine, IFN, tamoxifen, IL-2, GM-CSF, or a combination thereof, to a patient having scleroderma or cutaneous vasculitis.

In one aspect, the methods provided herein comprise administering to a patient with MDS a compound D formulation in combination with azacitidine, cytarabine, daunomycin, decitabine, idarubicin, lenalidomide, enzidipine, or a combination thereof.

In one aspect, the methods provided herein comprise administering compound D in combination with one or more second agents selected from the group consisting of a JAK inhibitor, a FLT3 inhibitor, an mTOR inhibitor, a spliceosome inhibitor, a BET inhibitor, an SMG1 inhibitor, an ERK inhibitor, a LSD1 inhibitor, a BH3 mimetic, a topoisomerase inhibitor, and a RTK inhibitor to a patient having a hematologic cancer. In one aspect, the methods provided herein comprise administering to a patient having a hematologic cancer a compound D formulation in combination with one or more second agents selected from a JAK inhibitor, a FLT3 inhibitor, an mTOR inhibitor, a spliceosome inhibitor, a BET inhibitor, an SMG1 inhibitor, an ERK inhibitor, a LSD1 inhibitor, a BH3 mimetic, a topoisomerase inhibitor, and a RTK inhibitor.

In one aspect, the methods provided herein comprise administering compound D in combination with one or more second agents selected from a JAK inhibitor, an FLT3 inhibitor, an mTOR inhibitor, a spliceosome inhibitor, a BET inhibitor, an SMG1 inhibitor, an ERK inhibitor, an LSD1 inhibitor, a BH3 mimetic, a topoisomerase inhibitor, and an RTK inhibitor to a patient having leukemia. In certain embodiments, compound D formulations provided herein are administered to a patient having leukemia in combination with one or more second agents selected from JAK inhibitors, FLT3 inhibitors, mTOR inhibitors, spliceosome inhibitors, BET inhibitors, SMG1 inhibitors, ERK inhibitors, LSD1 inhibitors, BH3 mimetics, topoisomerase inhibitors, and RTK inhibitors.

In one aspect, the methods provided herein comprise administering compound D in combination with one or more second agents selected from the group consisting of a JAK inhibitor, a FLT3 inhibitor, an mTOR inhibitor, a spliceosome inhibitor, a BET inhibitor, an SMG1 inhibitor, an ERK inhibitor, a LSD1 inhibitor, a BH3 mimetic, a topoisomerase inhibitor, and a RTK inhibitor to a patient with AML. In certain embodiments, compound D formulations provided herein are administered to a patient with AML in combination with one or more second agents selected from the group consisting of JAK inhibitors, FLT3 inhibitors, mTOR inhibitors, spliceosome inhibitors, BET inhibitors, SMG1 inhibitors, ERK inhibitors, LSD1 inhibitors, BH3 mimetics, topoisomerase inhibitors, and RTK inhibitors.

In one aspect, the methods provided herein comprise administering compound D in combination with an mTOR inhibitor to a patient having leukemia. In certain embodiments, compound D formulations provided herein are administered to a patient having leukemia in combination with an mTOR inhibitor. In certain embodiments, the mTOR inhibitor is selected from everolimus, MLN-0128, and AZD 8055. In some embodiments, the mTOR inhibitor is an mTOR kinase inhibitor. In certain embodiments, the mTOR kinase inhibitor is selected from 7- (6- (2-hydroxypropyl-2-yl) pyridin-3-yl) -1- ((trans) -4-methoxycyclohexyl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one (CC-223) and 1-ethyl-7- (2-methyl-6- (1H-1,2, 4-triazol-3-yl) pyridin-3-yl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one (CC-115). In certain embodiments, compound D is administered to a patient suffering from leukemia in combination with 7- (6- (2-hydroxypropyl-2-yl) pyridin-3-yl) -1- ((trans) -4-methoxycyclohexyl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one (CC-223). In certain embodiments, compound D is administered to a patient having leukemia in combination with 1-ethyl-7- (2-methyl-6- (1H-1,2, 4-triazol-3-yl) pyridin-3-yl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one (CC-115). In certain embodiments, compound D is administered to a patient having leukemia in combination with everolimus. In certain embodiments, compound D is administered to a patient with leukemia in combination with MLN-0128. In certain embodiments, compound D is administered to a patient having leukemia in combination with AZD 8055.

In one aspect, the methods provided herein comprise administering a combination of compound D and an mTOR inhibitor to a patient with AML. In certain embodiments, compound D formulations provided herein are administered to patients with AML in combination with an mTOR inhibitor. In certain embodiments, the mTOR inhibitor is selected from everolimus, MLN-0128, and AZD 8055. In some embodiments, the mTOR inhibitor is an mTOR kinase inhibitor. In certain embodiments, the mTOR kinase inhibitor is selected from 7- (6- (2-hydroxypropyl-2-yl) pyridin-3-yl) -1- ((trans) -4-methoxycyclohexyl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one (CC-223) and 1-ethyl-7- (2-methyl-6- (1H-1,2, 4-triazol-3-yl) pyridin-3-yl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one (CC-115). In certain embodiments, compound D is administered to a patient with AML in combination with 1-ethyl-7- (2-methyl-6- (1H-1,2, 4-triazol-3-yl) pyridin-3-yl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one. In certain embodiments, compound D is administered to a patient with AML in combination with everolimus. In certain embodiments, everolimus is administered to a patient with AML prior to administration of compound D. In certain embodiments, compound D is administered to patients with AML in combination with MLN-0128. In certain embodiments, compound D is administered to a patient with AML in combination with AZD 8055.

In one aspect, the methods provided herein comprise administering compound D in combination with a JAK inhibitor to a patient having MPN. In certain embodiments, the compound D formulations provided herein are administered to a patient with MPN in combination with a JAK inhibitor. In one aspect, the JAK inhibitor is selected from a JAK1 inhibitor, a JAK2 inhibitor, and a JAK3 inhibitor. In certain embodiments, the JAK inhibitor is selected from tofacitinib, morronib, nonglutib, decernotinib, barretinib (barcetitinib), ruxolitinib (ruxolitinib), feitinib (fedratinib), NS-018, and pacitinib (pacritinib). In certain embodiments, the JAK inhibitor is selected from tofacitinib, molotinib, ruxolitinib, phenanthratinib, NS-018, and pactinib. In certain embodiments, compound D is administered to a patient having MPN in combination with tofacitinib. In certain embodiments, compound D is administered to a patient having MPN in combination with molonetinib. In certain embodiments, compound D is administered to a patient suffering from MPN in combination with non-golitinib. In certain embodiments, compound D is administered to a patient having MPN in combination with decernotiib. In certain embodiments, compound D is administered to a patient having MPN in combination with baricetinib. In certain embodiments, compound D is administered to a patient having MPN in combination with ruxolitinib. In certain embodiments, compound D is administered to a patient having MPN in combination with phenanthratinib. In certain embodiments, compound D is administered to a patient having MPN in combination with NS-018. In certain embodiments, compound D is administered to a patient having MPN in combination with paktinib. In certain embodiments, MPN is independent of IL-3. In certain embodiments, the MPN is characterized by a JAK2 mutation, e.g., JAK2V617F mutation.

In one aspect, the methods provided herein comprise administering compound D in combination with a JAK inhibitor to a patient having myelofibrosis. In certain embodiments, compound D formulations provided herein are administered to a patient having myelofibrosis in combination with a JAK inhibitor. In one aspect, the JAK inhibitor is selected from a JAK1 inhibitor, a JAK2 inhibitor, and a JAK3 inhibitor. In certain embodiments, the JAK inhibitor is selected from tofacitinib, molotinib, ruxolitinib, phenanthratinib, NS-018, and pactinib. In certain embodiments, compound D is administered to a patient having myelofibrosis in combination with tofacitinib. In certain embodiments, compound D is administered to a patient having myelofibrosis in combination with molonetinib. In certain embodiments, compound D is administered to a patient having myelofibrosis in combination with ruxolitinib. In certain embodiments, compound D is administered to a patient having myelofibrosis in combination with phenanthratinib. In certain embodiments, compound D is administered to a patient having myelofibrosis in combination with NS-018. In certain embodiments, compound D is administered to a patient having myelofibrosis in combination with paktinib. In certain embodiments, myelofibrosis is characterized by a JAK2 mutation, for example, the JAK2V617F mutation. In some embodiments, the myelofibrosis is primary myelofibrosis. In other embodiments, the myelofibrosis is secondary myelofibrosis. In some such embodiments, the secondary myelofibrosis is post-polycythemia vera myelofibrosis. In other embodiments, the secondary myelofibrosis is myelofibrosis following essential thrombocythemia.

In one aspect, the methods provided herein comprise administering compound D in combination with a JAK inhibitor to a patient having leukemia. In certain embodiments, the compound D formulations provided herein are administered to a patient having leukemia in combination with a JAK inhibitor. In one aspect, the JAK inhibitor is selected from a JAK1 inhibitor, a JAK2 inhibitor, and a JAK3 inhibitor. In certain embodiments, the JAK inhibitor is selected from tofacitinib, molotinib, fegolitinib, descerntotinib, baricitinib, ruxolitinib, felatinib, NS-018, and pactinib. In certain embodiments, the JAK inhibitor is selected from the group consisting of molutinib, ruxolitinib, phenanthratinib, NS-018, and pactinib. In certain embodiments, compound D is administered to a patient having leukemia in combination with tofacitinib. In certain embodiments, compound D is administered to a patient having leukemia in combination with molonetinib. In certain embodiments, compound D is administered to a patient having leukemia in combination with non-golitinib. In certain embodiments, compound D is administered to a patient having leukemia in combination with decernotiib. In certain embodiments, compound D is administered to a patient having leukemia in combination with baricitinib. In certain embodiments, compound D is administered to a patient having leukemia in combination with ruxolitinib. In certain embodiments, compound D is administered to a patient having leukemia in combination with phenanthratinib. In certain embodiments, compound D is administered to a patient having leukemia in combination with NS-018. In certain embodiments, compound D is administered to a patient having leukemia in combination with paktinib. In certain embodiments, the MPN is characterized by a JAK2 mutation, e.g., JAK2V617F mutation.

In one aspect, the methods provided herein comprise administering compound D in combination with a JAK inhibitor to a patient having AML. In certain embodiments, the compound D formulations provided herein are administered to a patient with AML in combination with a JAK inhibitor. In one aspect, the JAK inhibitor is selected from a JAK1 inhibitor, a JAK2 inhibitor, and a JAK3 inhibitor. In certain embodiments, the JAK inhibitor is selected from tofacitinib, molotinib, fegolitinib, descerntotinib, baricitinib, ruxolitinib, felatinib, NS-018, and pactinib. In certain embodiments, the JAK inhibitor is selected from the group consisting of molutinib, ruxolitinib, phenanthratinib, NS-018, and pactinib. In certain embodiments, compound D is administered to a patient with AML in combination with tofacitinib. In certain embodiments, compound D is administered to a patient with AML in combination with molonetinib. In certain embodiments, compound D is administered in combination with non-golitinib to a patient suffering from AML. In certain embodiments, compound D is administered to a patient with AML in combination with decernotiib. In certain embodiments, compound D is administered to a patient with AML in combination with baricitinib. In certain embodiments, compound D is administered to a patient with AML in combination with ruxolitinib. In certain embodiments, compound D is administered to a patient with AML in combination with phenanthratinib. In certain embodiments, compound D is administered to a patient with AML in combination with NS-018. In certain embodiments, compound D is administered to a patient with AML in combination with paktinib. In certain embodiments, the MPN is characterized by a JAK2 mutation, e.g., JAK2V617F mutation.

In one aspect, the methods provided herein comprise administering compound D in combination with a FLT3 kinase inhibitor to a patient having leukemia. In certain embodiments, compound D formulations provided herein are administered to a patient having leukemia in combination with a FLT3 kinase inhibitor. In certain embodiments, the FLT3 kinase inhibitor is selected from the group consisting of quinitinib, sunitinib malate, midostaurin, pexidinib, lestaurtinib, tandutinib, and crenolanib. In certain embodiments, compound D is administered to a patient having leukemia in combination with quinazatinib. In certain embodiments, compound D is administered to a patient having leukemia in combination with sunitinib. In certain embodiments, compound D is administered to a patient suffering from leukemia in combination with midostaurin. In certain embodiments, compound D is administered to a patient having leukemia in combination with pexidasatinib. In certain embodiments, compound D is administered to a patient having leukemia in combination with lestaurtinib. In certain embodiments, compound D is administered to a patient having leukemia in combination with tandutinib. In certain embodiments, compound D is administered to a patient having leukemia in combination with crenolanib. In certain embodiments, the patient carries the FLT3-ITD mutation.

In one aspect, the methods provided herein comprise administering compound D in combination with a FLT3 kinase inhibitor to a patient with AML. In certain embodiments, the compound D formulations provided herein are administered to a patient with AML in combination with a FLT3 kinase inhibitor. In certain embodiments, the FLT3 kinase inhibitor is selected from the group consisting of azatinib, sunitinib malate, midostaurin, pexidinib, lestaurtinib, tandutinib, azatinib, and crenolanib. In certain embodiments, compound D is administered to a patient with AML in combination with quinazatinib. In certain embodiments, compound D is administered to a patient having AML in combination with sunitinib. In certain embodiments, compound D is administered to a patient suffering from AML in combination with midostaurin. In certain embodiments, compound D is administered to a patient with AML in combination with pexidinib. In certain embodiments, compound D is administered to a patient with AML in combination with lestaurtinib. In certain embodiments, compound D is administered to a patient with AML in combination with tandutinib. In certain embodiments, compound D is administered to a patient with AML in combination with crenolanib. In certain embodiments, the patient carries the FLT3-ITD mutation.

In certain embodiments, compound D is administered to a patient having leukemia in combination with a spliceosome inhibitor. In certain embodiments, compound D is administered to a patient with AML in combination with a spliceosome inhibitor. In certain embodiments, the spliceosome inhibitor is pladienolide B (pladienolide B), 6-deoxypladienolide D, or H3B-8800.

In one aspect, the methods provided herein comprise administering compound D in combination with an SMG1 kinase inhibitor to a patient having leukemia. In certain embodiments, the compound D formulations provided herein are administered to a patient having leukemia in combination with an SMG1 kinase inhibitor. In one aspect, the methods provided herein comprise administering compound D in combination with an SMG1 kinase inhibitor to a patient having AML. In certain embodiments, the compound D formulations provided herein are administered to a patient with AML in combination with an SMG1 kinase inhibitor. In certain embodiments, the SMG1 inhibitor is 1-ethyl-7- (2-methyl-6- (1H-1,2, 4-triazol-3-yl) pyridin-3-yl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one, chloro-N, N-diethyl-5- ((4- (2- (4- (3-methylureido) phenyl) pyridin-4-yl) pyrimidin-2-yl) amino) benzenesulfonamide (compound Ii) or a compound disclosed in a.gopalsmy et al, bioorg.med Chem lett.2012,22:6636- Pyrimidin-2-yl) amino) benzenesulfonamides.

In one aspect, the methods provided herein comprise administering compound D in combination with a BCL2 inhibitor to a patient having leukemia. In certain embodiments, the compound D formulations provided herein are administered to a patient having leukemia in combination with a BCL2 inhibitor. In certain embodiments, compound D is administered to a patient with AML in combination with a BCL2 inhibitor. In certain embodiments, compound D formulations provided herein are administered to a patient with AML in combination with a BCL2 inhibitor (e.g., venetock or navitoclax). In certain embodiments, the BCL2 inhibitor is teneptogram.

In one embodiment, provided herein is a method for treating AML resistant to treatment with a BCL2 inhibitor, comprising administering compound D. In one embodiment, provided herein is a method for treating AML with acquired resistance to venetork treatment comprising administering compound D. In one embodiment, provided herein is a method for treating AML with acquired resistance to vinatock treatment comprising administering a combination of compound D and a BCL2 inhibitor. In one embodiment, provided herein is a method for treating AML with acquired resistance to teneptogram treatment comprising administering a combination of compound D and teneptogram.

In one aspect, the methods provided herein comprise administering compound D in combination with a topoisomerase inhibitor to a patient having leukemia. In certain embodiments, the compound D formulations provided herein are administered to a patient having leukemia in combination with a topoisomerase inhibitor. In certain embodiments, compound D is administered to a patient with AML in combination with a topoisomerase inhibitor. In certain embodiments, compound D formulations provided herein are administered to a patient suffering from AML in combination with a topoisomerase inhibitor, e.g., irinotecan, topotecan, camptothecin, lamellarin D, etoposide, teniposide, doxorubicin, daunomycin, mitoxantrone, amsacrine, ellipticine (elliticine), aurintricarboxylic acid, or HU-331. In certain embodiments, the topoisomerase inhibitor is topotecan.

In certain embodiments, compound D is administered to a patient having leukemia in combination with a BET inhibitor. In certain embodiments, compound D is administered to a patient with AML in combination with a BET inhibitor. In certain embodiments, the BET inhibitor is selected from GSK525762A, OTX015, BMS-986158, TEN-010, CPI-0610, INCB54329, BAY1238097, FT-1101, C90010, ABBV-075, BI 894999, GS-5829, GSK1210151A (I-BET-151), CPI-203, RVX 208, XD46, MS436, PFI-1, RVX2135, ZEN3365, XD14, ARV-771, MZ-1, PLX5117, 4- [2- (cyclopropylmethoxy) -5- (methylsulfonyl) phenyl ] -2-methylisoquinolin-1 (2H) -one (Compound A), EP11313, and EP 11336.

In certain embodiments, compound D is administered to a patient having leukemia in combination with a LSD1 inhibitor. In certain embodiments, compound D is administered to a patient with AML in combination with a LSD1 inhibitor. In certain embodiments, the LSD1 inhibitor is selected from the group consisting of ary-1001, ary-2001, INCB-59872, IMG-7289, TAK 418, GSK-2879552, and 4- [2- (4-amino-piperidin-1-yl) -5- (3-fluoro-4-methoxy-phenyl) -1-methyl-6-oxo-1, 6-dihydropyrimidin-4-yl ] -2-fluoro-benzonitrile or a salt thereof (e.g., benzenesulfonate, compound B).

In one aspect, the methods provided herein comprise administering compound D to a patient having leukemia with triptolide, retastatin (retastatin), adriamycin (alvemycin), 7- (6- (2-hydroxypropyl-2-yl) pyridin-3-yl) -1- ((trans) -4-methoxycyclohexyl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one (CC-223), 1-ethyl-7- (2-methyl-6- (1H-1,2, 4-triazol-3-yl) pyridin-3-yl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one (CC-115), A combination of rapamycin, MLN-0128, everolimus, AZD8055, pladienolide B, topotecan, thioguanine, mitoxantrone, etoposide, decitabine, daunomycin, clofarabine, cladribine, 6-mercaptopurine, chloro-N, N-diethyl-5- ((4- (2- (4- (3-methylureido) phenyl) pyridin-4-yl) pyrimidin-2-yl) amino) benzenesulfonamide (compound Ii), fidatinib, sunitinib, pexitinib, midostaurin, lestatinib, morlotinib, quinatinib and encalonib.

In one aspect, the methods provided herein comprise administering compound D with triptolide, retamycin, apramycin, 7- (6- (2-hydroxypropyl-2-yl) pyridin-3-yl) -1- ((trans) -4-methoxycyclohexyl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one (CC-223), 1-ethyl-7- (2-methyl-6- (1H-1,2, 4-triazol-3-yl) pyridin-3-yl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one (CC-115), to a patient having AML, A combination of rapamycin, MLN-0128, everolimus, AZD8055, pladienolide B, topotecan, thioguanine, mitoxantrone, etoposide, decitabine, daunomycin, clofarabine, cladribine, 6-mercaptopurine, chloro-N, N-diethyl-5- ((4- (2- (4- (3-methylureido) phenyl) pyridin-4-yl) pyrimidin-2-yl) amino) benzenesulfonamide (compound Ii), fidatinib, sunitinib, pexitinib, midostaurin, lestatinib, morlotinib, quinatinib and encalonib.

In one aspect, the methods provided herein comprise administering compound D in combination with an mTOR inhibitor to a patient having a cancer, wherein the cancer is selected from the group consisting of breast cancer, renal cancer, pancreatic cancer, gastrointestinal cancer, lung cancer, neuroendocrine tumor (NET), and Renal Cell Carcinoma (RCC). In certain embodiments, the compound D formulations provided herein are administered to a patient having cancer in combination with a topoisomerase inhibitor. In certain embodiments, compound D formulations provided herein are administered to a cancer patient in combination with an mTOR inhibitor, wherein the cancer is selected from the group consisting of breast cancer, renal cancer, pancreatic cancer, gastrointestinal cancer, lung cancer, neuroendocrine tumor (NET), and renal cell carcinoma. In certain embodiments, the mTOR inhibitor is selected from everolimus, MLN-0128, and AZD 8055. In some embodiments, the mTOR inhibitor is an mTOR kinase inhibitor. In certain embodiments, the mTOR kinase inhibitor is selected from 7- (6- (2-hydroxypropyl-2-yl) pyridin-3-yl) -1- ((trans) -4-methoxycyclohexyl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one (CC-223) and 1-ethyl-7- (2-methyl-6- (1H-1,2, 4-triazol-3-yl) pyridin-3-yl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one (CC-115). In one embodiment, the mTOR kinase inhibitor is 7- (6- (2-hydroxypropyl-2-yl) pyridin-3-yl) -1- ((trans) -4-methoxycyclohexyl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one (CC-223). In one embodiment, the mTOR kinase inhibitor is 1-ethyl-7- (2-methyl-6- (1H-1,2, 4-triazol-3-yl) pyridin-3-yl) -3, 4-dihydropyrazino [2,3-b ] pyrazin-2 (1H) -one (CC-115). In one embodiment, the mTOR inhibitor is everolimus. In one embodiment, the mTOR inhibitor is temsirolimus. In one embodiment, the mTOR inhibitor is MLN-0128. In one embodiment, the mTOR inhibitor is AZD 8055.

In certain embodiments, compound D is administered to a breast cancer patient in combination with everolimus. In certain embodiments, compound D formulations provided herein are administered to a breast cancer patient in combination with everolimus.

In certain embodiments, compound D is administered to a patient with renal cancer in combination with everolimus. In certain embodiments, compound D formulations provided herein are administered to a renal cancer patient in combination with everolimus.

In certain embodiments, compound D is administered to a pancreatic cancer patient in combination with everolimus. In certain embodiments, compound D formulations provided herein are administered to a pancreatic cancer patient in combination with everolimus.

In certain embodiments, compound D is administered to a gastrointestinal cancer patient in combination with everolimus. In certain embodiments, compound D formulations provided herein are administered to a gastrointestinal cancer patient in combination with everolimus.

In certain embodiments, compound D is administered to a lung cancer patient in combination with everolimus. In certain embodiments, compound D formulations provided herein are administered to a lung cancer patient in combination with everolimus.

In certain embodiments, compound D is administered to a neuroendocrine tumor patient in combination with everolimus. In certain embodiments, compound D formulations provided herein are administered to a neuroendocrine tumor patient in combination with everolimus.

In certain embodiments, compound D is administered to a renal cell carcinoma patient in combination with everolimus. In certain embodiments, compound D formulations provided herein are administered to a renal cell carcinoma patient in combination with everolimus.

Also contemplated herein is a method of increasing the dose of an anti-cancer drug or agent that can be safely and effectively administered to a patient, the method comprising administering to a patient (e.g., a human) compound D (e.g., a compound D formulation provided herein) in combination with a second anti-cancer drug. Patients that may benefit by this method are those who may suffer from adverse effects associated with anticancer drugs used to treat specific cancers of skin cancer, subcutaneous tissue cancer, lymph node cancer, brain cancer, lung cancer, liver cancer, bone cancer, intestinal cancer, colon cancer, heart cancer, pancreatic cancer, adrenal cancer, kidney cancer, prostate cancer, breast cancer, colorectal cancer, or combinations thereof. Administration of compound D (e.g., the compound D formulations provided herein) mitigates or alleviates the severity of adverse effects such that it would otherwise limit the amount of anti-cancer drug.

Also contemplated herein is a method of reducing the dose of an anti-cancer drug or agent that can be safely and effectively administered to a patient, the method comprising administering to a patient (e.g., a human) compound D (e.g., a compound D formulation provided herein) in combination with a second anti-cancer drug. Patients that may benefit by this method are those who may suffer from adverse effects associated with anticancer drugs used to treat specific cancers of skin cancer, subcutaneous tissue cancer, lymph node cancer, brain cancer, lung cancer, liver cancer, bone cancer, intestinal cancer, colon cancer, heart cancer, pancreatic cancer, adrenal cancer, kidney cancer, prostate cancer, breast cancer, colorectal cancer, or combinations thereof. Administration of compound D (e.g., compound D formulations provided herein) enhances the activity of the anticancer drug, which allows for dose reduction of the anticancer drug while maintaining efficacy, which in turn can mitigate or lessen the severity of adverse effects such that it limits the anticancer drug amount.

In one embodiment, compound D is administered daily in an amount in the range of about 0.1 to about 20mg, about 1 to about 15mg, about 1 to about 10mg, or about 1 to about 15mg before, during, or after the occurrence of an adverse effect associated with administration of an anti-cancer drug to a patient. In certain embodiments, compound D is administered in combination with a specific agent (such as heparin, aspirin, coumarin, or G-CSF) to avoid adverse effects associated with anti-cancer drugs, such as, but not limited to, neutropenia or thrombocytopenia.

In one embodiment, compound D (e.g., a compound D formulation provided herein) is administered to a patient suffering from a disease or disorder associated with or characterized by undesired angiogenesis in combination with additional active ingredients including, but not limited to, anti-cancer agents, anti-inflammatory agents, antihistamines, antibiotics, and steroids.

In another embodiment, encompassed herein is a method of treating, preventing and/or managing cancer, comprising administering compound D (e.g., a compound D formulation provided herein) in combination with (e.g., before, during or after) at least one anti-cancer therapy, including but not limited to surgery, immunotherapy, biologic therapy, radiation therapy or other non-drug based therapies currently used to treat, prevent and/or manage cancer. The use of the compounds provided herein in combination with other anti-cancer therapies can provide unique treatment regimens that are unexpectedly effective in certain patients. Without being limited by theory, it is believed that compound D may provide an additive or synergistic effect when administered concurrently with at least one anti-cancer therapy.

As discussed elsewhere herein, a method of reducing, treating, and/or preventing adverse or unwanted effects associated with other anti-cancer therapies is contemplated herein, including, but not limited to, surgery, chemotherapy, radiation therapy, hormonal therapy, biological therapy, and immunotherapy. Compound D (e.g., compound D formulations provided herein) and other active ingredients can be administered to a patient before, during, or after adverse effects associated with other anti-cancer therapies occur.

In certain embodiments, the methods provided herein comprise administering compound D with one or more of calcium, calcitriol, or vitamin D supplements. In certain embodiments, the methods provided herein comprise administering calcium, calcitriol, and a vitamin D supplement prior to treatment with compound D. In certain embodiments, the methods provided herein comprise administering calcium, calcitriol, and a vitamin D supplement prior to administering the first dose of compound D in each cycle. In certain embodiments, the methods provided herein comprise administering calcium, calcitriol, and vitamin D supplement at least up to 3 days prior to treatment with compound D. In certain embodiments, the methods provided herein comprise administering calcium, calcitriol, and a vitamin D supplement prior to administering the first dose of compound D in each cycle. In certain embodiments, the methods provided herein comprise administering calcium, calcitriol, and vitamin D supplement at least up to 3 days prior to administering the first dose of compound D in each cycle. In certain embodiments, the methods provided herein comprise administering calcium, calcitriol, and a vitamin D supplement prior to administering the first dose of compound D in each cycle and continuing after administering the last dose of compound D in each cycle. In certain embodiments, the methods provided herein comprise administering calcium, calcitriol, and vitamin D supplement at least up to 3 days prior to administering the first dose of compound D in each cycle and continuing until at least up to 3 days after administering the last dose of compound D in each cycle (e.g., at least up to day 8 when compound D is administered on days 1-5). In one embodiment, the methods provided herein comprise administering the calcium, calcitriol, and vitamin D supplement at least up to 3 days prior to administration on day 1 of each cycle and continuing until ≧ 3 days after the last dose of compound D in each cycle (e.g., ≧ day 8 when compound D is administered on days 1-5, and ≧ day 13 when compound D is administered on days 1-3 and 8-10).

In certain embodiments, the calcium supplement is administered to deliver at least 1200mg of elemental calcium per day, divided into multiple doses. In certain embodiments, the calcium supplement is administered in the form of calcium carbonate at a dose of 500mg, orally (PO) three times daily.

In certain embodiments, the calcitriol supplement is administered to deliver 0.25 μ g of calcitriol (PO) once per day.

In certain embodiments, the vitamin D supplement is administered to deliver from about 500IU to about 50,000IU of vitamin D once per day. In certain embodiments, the vitamin D supplement is administered to deliver about 1000IU of vitamin D once per day. In certain embodiments, the vitamin D supplement is administered to deliver about 50,000IU of vitamin D once per week. In certain embodiments, the vitamin D supplement is administered to deliver about 1000IU of vitamin D2 or D3 once a day. In certain embodiments, the vitamin D supplement is administered to deliver about 500IU of vitamin D once a day. In certain embodiments, the vitamin D supplement is administered to deliver about 50,000IU of vitamin D once per week. In certain embodiments, the vitamin D supplement is administered to deliver about 20,000IU of vitamin D once per week. In certain embodiments, the vitamin D supplement is administered to deliver about 1000IU of vitamin D2 or D3 once a day. In certain embodiments, the vitamin D supplement is administered to deliver about 50,000IU of vitamin D2 or D3 once per week. In certain embodiments, the vitamin D supplement is administered to deliver about 20,000IU of vitamin D2 or D3 once per week.

In certain embodiments, the compound D formulations provided herein and docetaxel are administered to a patient having non-small cell lung cancer who was previously treated with carbo/VP 16 and radiation therapy.

Use with transplantation therapy

Compound D (e.g., the compound D formulations provided herein) can be used to reduce the risk of Graft Versus Host Disease (GVHD). Accordingly, encompassed herein is a method of treating, preventing and/or managing cancer, comprising administering compound D (e.g., a compound D formulation provided herein) in conjunction with transplantation therapy.

As is known to those of ordinary skill in the art, treatment of cancer is often based on the stage and mechanism of the disease. For example, with inevitable leukemic transformation occurring at some stages of cancer, transplantation of peripheral blood stem cells, hematopoietic stem cell preparations or bone marrow may be required. The use of compound D (e.g., the compound D formulations provided herein) in combination with transplantation therapy provides a unique and unexpected synergistic effect. In particular, the compound D formulations provided herein exhibit immunomodulatory activity, which may provide additive or synergistic effects when given concurrently with transplantation therapy of patients with cancer.

Compound D (e.g., the compound D formulations provided herein) can be used in combination with transplantation therapy, thereby reducing the risk of complications and GVHD associated with invasive procedures of transplantation. Encompassed herein is a method of treating, preventing and/or managing cancer, comprising administering a compound D formulation provided herein to a patient (e.g., a human) prior to, during or after transplanting umbilical cord blood, placental blood, peripheral blood stem cells, hematopoietic stem cell preparation or bone marrow. Some examples of stem cells suitable for use in the methods provided herein are disclosed in U.S. patent No. 7,498,171, the disclosure of which is incorporated by reference herein in its entirety.

In one embodiment, compound D (e.g., a compound D formulation provided herein) is administered to a patient having acute myeloid leukemia before, during, or after transplantation.

In one embodiment, compound D (e.g., compound D formulations provided herein) is administered to a patient with multiple myeloma before, during, or after autologous peripheral blood progenitor cell transplantation.

In one embodiment, compound D (e.g., compound D formulations provided herein) is administered to a patient with NHL (e.g., DLBCL) before, during, or after autologous peripheral blood progenitor cell transplantation.

Periodic therapy

In certain embodiments, compound D (e.g., a compound D formulation provided herein) is periodically administered to a patient independently of the cancer being treated. Periodic therapy involves administering the active agent for a period of time, then discontinuing the administration for a period of time, and repeating this sequential administration. Periodic therapy can reduce the development of resistance to one or more therapies, avoid or reduce the adverse effects of a therapy, and/or improve the efficacy of a treatment.

In certain embodiments, compound D (e.g., a compound D formulation provided herein) is administered daily in single or divided doses over a period of four to six weeks with a rest period of about one or two weeks. In certain embodiments, compound D (e.g., a compound D formulation provided herein) is administered daily in single or divided doses for one to ten consecutive days of a 28-day cycle, followed by a drug-off period of no administration for the remainder of the 28-day cycle. The periodic method further allows for increasing the frequency, number and length of administration cycles. Thus, in certain embodiments, contemplated herein is the application of compound D (e.g., a compound D formulation provided herein) which cycles more than it typically would be when applied alone. In certain embodiments, administration of compound D (e.g., the compound D formulations provided herein) in greater number of cycles will typically cause dose-limiting toxicity in patients who have also not been administered a second active ingredient.

In one embodiment, compound D (e.g., a compound D formulation provided herein) is administered daily and continuously for three or four weeks to administer a dose of about 0.1 to about 20mg/D of compound D, followed by one or two weeks of discontinuation.

In another embodiment, compound D (e.g., a compound D formulation provided herein) is administered intravenously, and the second active ingredient is administered orally, wherein administration of compound D (e.g., a compound D formulation provided herein) is performed 30 to 60 minutes prior to the second active ingredient over a period of four to six weeks. In certain embodiments, the combination of compound D (e.g., compound D formulations provided herein) and the second active ingredient is administered by intravenous infusion within about 90 minutes of each cycle. In certain embodiments, a cycle comprises administering about 0.1 to about 150 mg/day of compound D (e.g., a compound D formulation provided herein) and about 50 to about 200mg/m 2/day of the second active ingredient daily for three to four weeks and then discontinuing the administration for one to two weeks. In certain embodiments, the number of cycles of administering the combination therapy to the patient ranges from about one to about 24 cycles, about two to about 16 cycles, or about four to about three cycles.

In one embodiment, the periodic therapy provided herein comprises administration of compound D (e.g., a compound D formulation provided herein) in a treatment cycle comprising an administration period of up to 5 days followed by a drug withdrawal period. In one embodiment, the treatment cycle comprises an administration period of 5 days followed by a withdrawal period. In one embodiment, the treatment cycle comprises an administration period of up to 10 days followed by a drug withdrawal period. In one embodiment, the drug withdrawal period is from about 10 days to about 40 days. In one embodiment, the treatment cycle includes an administration period of up to 10 days followed by a drug withdrawal period of about 10 days to about 40 days. In one embodiment, the treatment cycle includes an administration period of up to 10 days, followed by a drug withdrawal period of about 23 days to about 37 days. In one embodiment, the drug withdrawal period is from about 23 days to about 37 days. In one embodiment, the drug withdrawal period is 23 days. In one embodiment, the treatment cycle includes an administration period of up to 10 days followed by a 23 day rest period. In one embodiment, the drug withdrawal period is 37 days. In one embodiment, the treatment cycle comprises an administration period of up to 10 days followed by a 37 day rest period.

In one embodiment, the treatment cycle comprises administering compound D (e.g., a compound D formulation provided herein) on days 1 to 5 of a 28-day cycle. In another embodiment, the treatment cycle comprises administering compound D (e.g., a compound D formulation provided herein) on days 1 to 10 of a 28-day cycle. In one embodiment, the treatment cycle comprises administration on days 1 to 5 of a 42 day cycle. In another embodiment, the treatment cycle comprises administration on days 1 to 10 of a 42 day cycle. In another embodiment, the treatment cycle comprises administration on days 1 to 5 and 15 to 19 of a 28 day cycle. In another embodiment, the treatment cycle comprises administration on days 1 to 3 and 8 to 10 of a 28 day cycle.

In one embodiment, the treatment cycle comprises administering compound D (e.g., a compound D formulation provided herein) on days 1 to 21 of a 28-day cycle. In another embodiment, the treatment cycle comprises administration on days 1 to 5 of a 7 day cycle. In another embodiment, the treatment cycle comprises administration on days 1 to 7 of a 7 day cycle.

Any treatment cycle described herein may be repeated for at least 2, 3, 4, 5, 6, 7, 8 or more cycles. In certain instances, a treatment cycle as described herein includes 1 to about 24 cycles, about 2 to about 16 cycles, or about 2 to about 4 cycles. In certain instances, a treatment cycle as described herein comprises 1 to about 4 cycles. In certain embodiments, cycles 1 to 4 are all 28 day cycles. In certain embodiments, cycle 1 is a 42 day cycle, and cycles 2 to 4 are 28 day cycles. In some embodiments, compound D (e.g., a compound D formulation provided herein) is applied for 1 to 13 28 day cycles (e.g., about 1 year). In certain instances, periodic therapy is not limited to the number of cycles, and therapy continues until disease progression. In certain instances, a cycle may include varying the duration of the administration and/or withdrawal periods described herein.

In one embodiment, the treatment cycle comprises administering compound D at a dose of about 0.3 mg/day, 0.6 mg/day, 1.2 mg/day, 1.8 mg/day, 2.4 mg/day, 3.6 mg/day, 5.4 mg/day, 7.2 mg/day, 8.1 mg/day, 9.0 mg/day, 10.0 mg/day, 10.8 mg/day, or 12.2 mg/day, once daily. In one embodiment, the treatment cycle comprises administering compound D at a dose of about 0.3 mg/day, 0.6 mg/day, 1.2 mg/day, 1.8 mg/day, 2.4 mg/day, 3.6 mg/day, 5.4 mg/day, 7.2 mg/day, 8.1 mg/day, 9.0 mg/day, 10.0 mg/day, 10.8 mg/day, 12.2 mg/day, or 20 mg/day, once daily. In one embodiment, the treatment cycle comprises administering compound D at a dose of about 0.6 mg/day, 1.2 mg/day, 1.8 mg/day, 2.4 mg/day, or 3.6 mg/day, once daily. In some such embodiments, the treatment cycle comprises administering compound D at a dose of about 0.6mg, 1.2mg, 1.8mg, 2.4mg, or 3.6mg on days 1 to 3 of a 28-day cycle. In other embodiments, the treatment cycle comprises administering compound D at a dose of about 0.6mg, 1.2mg, 1.8mg, 2.4mg, or 3.6mg on days 1 through 5 and 15 through 19 of a 28 day cycle. In other embodiments, the treatment cycle comprises administering compound D at a dose of about 0.6mg, 1.2mg, 1.8mg, 2.4mg, 3.6mg, 5.4 mg/day, 7.2 mg/day, 8.1 mg/day, 9.0 mg/day, or 10.0 mg/day on days 1 through 5 and 15 through 19 of a 28-day cycle.

Compound D (e.g., a compound D formulation provided herein) can be administered in the same amount for all administration periods of one treatment cycle. Alternatively, in one embodiment, the compounds are administered at different doses during the administration.

In one embodiment, a compound D formulation provided herein is administered to a subject within a cycle, wherein the cycle comprises administering the formulation for at least 5 days of a 28 day cycle. In one embodiment, a compound D formulation provided herein is administered to a subject within a cycle, wherein the cycle comprises administering the formulation on days 1 to 5 of a 28 day cycle. In one embodiment, the formulation is applied to deliver a dose of compound D of about 0.1mg to about 20mg on days 1 to 5 of a 28 day cycle. In one embodiment, the formulation is applied to deliver a dose of compound D of about 0.5mg to about 5mg on days 1 to 5 of a 28 day cycle. In one embodiment, the formulation is applied to deliver a dose of compound D of about 0.5mg to about 10mg on days 1 to 5 of a 28 day cycle. In one embodiment, the compound D formulations provided herein are administered to a subject within a cycle, wherein the cycle comprises administering the formulation on days 1 to 5 and 15 to 19 of a 28 day cycle. In one embodiment, the formulation is applied to deliver a dose of compound D of about 0.1mg to about 20mg on days 1 to 5 and 15 to 19 of a 28 day cycle. In one embodiment, the formulation is applied to deliver a dose of compound D of about 0.5mg to about 5mg on days 1 to 5 and 15 to 19 of a 28 day cycle. In one embodiment, the formulation is applied to deliver a dose of compound D of about 0.5mg to about 10mg on days 1 to 5 and 15 to 19 of a 28 day cycle.

In one embodiment, provided herein is a method of treating AML by administering to a subject compound D provided herein within a cycle, wherein the cycle comprises administering a formulation to deliver a dose of compound D of about 0.1mg to about 20mg for at least 5 days of a 28-day cycle. In one embodiment, provided herein is a method of treating AML by administering to a subject compound D provided herein within a cycle, wherein the cycle comprises administering a formulation to deliver a dose of compound D of about 0.1mg to about 20mg on days 1 to 5 of a 28-day cycle. In one embodiment, provided herein is a method of treating AML by administering to a subject compound D provided herein within a cycle, wherein the cycle comprises administering a formulation to deliver a dose of compound D of about 0.1mg to about 5mg on days 1 to 5 of a 28-day cycle. In one embodiment, provided herein is a method of treating AML by administering to a subject compound D provided herein within a cycle, wherein the cycle comprises administering a formulation to deliver a dose of compound D of about 0.5mg to about 5mg on days 1 to 5 of a 28-day cycle. In another embodiment, provided herein is a method of treating AML by administering to a subject compound D provided herein within a cycle, wherein the cycle comprises administering a formulation to deliver a dose of compound D of about 0.1mg to about 20mg on days 1 to 5 and 15 to 19 of a 28-day cycle. In one embodiment, provided herein is a method of treating AML by administering to a subject compound D provided herein within a cycle, wherein the cycle comprises administering a formulation to deliver a dose of compound D of about 0.1mg to about 5mg on days 1 to 5 and 15 to 19 of a 28-day cycle. In one embodiment, provided herein is a method of treating AML by administering to a subject compound D provided herein within a cycle, wherein the cycle comprises administering a formulation to deliver a dose of compound D of about 0.5mg to about 5mg on days 1 to 5 and 15 to 19 of a 28-day cycle.

In one embodiment, provided herein is a method of treating MDS by administering compound D provided herein to a subject over a cycle, wherein the cycle comprises administering a formulation to deliver a dose of compound D of about 0.1mg to about 20mg for at least 5 days of a 28 day cycle. In one embodiment, provided herein is a method of treating MDS by administering compound D provided herein to a subject within a cycle, wherein the cycle comprises administering a formulation to deliver a dose of compound D of about 0.1mg to about 20mg on days 1 to 5 of a 28 day cycle. In one embodiment, provided herein is a method of treating MDS by administering compound D provided herein to a subject within a cycle, wherein the cycle comprises administering a formulation to deliver a dose of compound D of about 0.1mg to about 5mg on days 1 to 5 of a 28 day cycle. In one embodiment, provided herein is a method of treating MDS by administering compound D provided herein to a subject within a cycle, wherein the cycle comprises administering a formulation to deliver a dose of compound D of about 0.5mg to about 5mg on days 1 to 5 of a 28 day cycle. In another embodiment, provided herein is a method of treating MDS by administering compound D provided herein to a subject within a cycle, wherein the cycle comprises administering a formulation to deliver a dose of compound D of about 0.1mg to about 20mg on days 1 to 5 and 15 to 19 of a 28 day cycle. In one embodiment, provided herein is a method of treating MDS by administering compound D provided herein to a subject within a cycle, wherein the cycle comprises administering a formulation to deliver a dose of compound D of about 0.1mg to about 5mg on days 1 to 5 and 15 to 19 of a 28 day cycle. In one embodiment, provided herein is a method of treating MDS by administering compound D provided herein to a subject within a cycle, wherein the cycle comprises administering a formulation to deliver a dose of compound D of about 0.5mg to about 5mg on days 1 to 5 and 15 to 19 of a 28 day cycle.

5.3. Method for detecting and quantifying gene sets or biomarkers

In certain embodiments, provided herein are methods of detecting and quantifying RNA (e.g., mRNA) levels of a gene set (such as a gene signature or biomarker provided herein) from a biological sample. The methods of detecting and quantifying the mRNA levels of a gene set include any method known in the art that can detect or quantify mRNA, such as transcriptome profiling, quantitative RT-PCR (qRT-PCR), ribonuclease protection assays, Northern blotting, and the like.

Any suitable assay platform can be used to determine the presence of mRNA in a sample. For example, the assay may be in the form of a dipstick (dipstick), membrane, chip, disc, test strip, filter, microsphere, slide, multiwell plate or optical fiber. The assay system may have a solid support to which nucleic acids corresponding to mRNA are attached. Solid supports may include, for example, plastic, silicon, metal, resin, glass, membranes, particles, precipitates, gels, polymers, sheets, spheres, polysaccharides, capillaries, membranes, plates, or slides. Assay components can be prepared and packaged together as a kit for detecting mRNA.

If desired, the nucleic acid can be labeled to produce a population of labeled mRNA. In general, the sample can be labeled using methods well known in the art (e.g., using DNA ligase, terminal transferase, or by labeling RNA backbones, etc.). See, e.g., Ausubel et al, Short Protocols in Molecular Biology(Wiley&Sons, 3 rd edition 1995); the result of Sambrook et al,Molecular Cloning:A Laboratory Manual(Cold spring harbor, N.Y., 3 rd edition)2001). In some embodiments, the sample is labeled with a fluorescent label. Exemplary fluorescent dyes include, but are not limited to, xanthene dyes, fluorescein dyes (e.g., Fluorescein Isothiocyanate (FITC), 6-carboxyfluorescein (FAM), 6-carboxy-2 ', 4 ', 7 ', 4, 7-Hexachlorofluorescein (HEX), 6-carboxy-4 ', 5 ' -dichloro-2 ', 7 ' -dimethoxyfluorescein (JOE)), rhodamine dyes (e.g., rhodamine 110(R110), N, N ', N ' -tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-Rhodamine (ROX), 5-carboxyrhodamine 6G (R6G5 or G5), 6-carboxyrhodamine 6G (R6G6 or G6)), cyanine dyes (e.g., Cy3, Cy5 and Cy7), Alexa dyes (e.g., Alexa-fluor-555) Coumarins, diethylaminocoumarins, umbelliferones, benzoylimine dyes (e.g., Hoechst 33258), phenanthridine dyes (e.g., texas red), ethidium dyes, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, BODIPY dyes, quinoline dyes, pyrenes, chlorotriazinyl fluorescein, eosin dyes, tetramethylrhodamine, lissamine, naphthalocyanine and the like.

An example of a PCR method can be found in U.S. patent No. 6,927,024, which is incorporated herein by reference in its entirety. An example of an RT-PCR method can be found in U.S. Pat. No. 7,122,799, which is incorporated herein by reference in its entirety. Methods of fluorescence in situ PCR are described in U.S. Pat. No. 7,186,507, which is incorporated herein by reference in its entirety.

In some embodiments, qRT-PCR can be used for both detection and quantification of RNA targets (Bustin et al, Clin. Sci.2005,109: 365-. Quantitative results obtained by qRT-PCR generally provide more information than qualitative data. Thus, in some embodiments, qRT-PCR based assays can be used to measure mRNA levels during cell-based assays. The qRT-PCR method can also be used to monitor patient therapy. Examples of qRT-PCR based methods can be found, for example, in U.S. patent No. 7,101,663, which is incorporated herein by reference in its entirety. Instruments for qRT-PCR (such as Applied Biosystems 7500) are commercially available, as are reagents, such as

Sequence Detection Chemistry. For example, it can be used according to the manufacturer's instructions

Gene Expression Assays. These kits are pre-formulated gene expression assays for rapid, reliable detection and quantification of human, mouse and rat mRNA transcripts. For example, an exemplary qRT-PCR procedure is 50 ℃ for 2 minutes, 95 ℃ for 10 minutes, 40 cycles of 95 ℃ for 15 seconds, then 60 ℃ for 1 minute.

To determine that the fluorescence signal associated with a particular amplicon accumulation crosses a threshold (referred to as C) _T ) Can analyze the data, e.g., using 7500 real-time PCR System sequence detection software versus using comparative C _T Relative quantitative calculation method. Using this method, the output is expressed as fold change in expression level. In some embodiments, the threshold level may optionally be automatically determined by software. In some embodiments, the threshold level is set above baseline, but low enough to be within the exponentially growing region of the amplification curve.

In some embodiments, provided herein are methods of detecting and quantifying cDNA levels of a gene set (such as a gene signature or biomarker provided herein) from a biological sample. In certain embodiments, the method further comprises generating cDNA from mRNA obtained from the sample. Any method known in the art for producing cDNA from mRNA can be used herein. Methods of detecting and quantifying the cDNA level of a gene set include any method known in the art that can detect or quantify cDNA, such as DNA microarrays, high throughput sequencing, Southern blots, and the like.

In some embodiments, provided herein are methods of detecting and quantifying protein levels of a gene set (such as a gene signature or biomarker provided herein) from a biological sample. The methods of detecting and quantifying the protein levels of a gene set include any method known in the art that can detect or quantify proteins, such as mass spectrometry, immunohistochemistry, flow cytometry, cytometric bead arrays, ELISA, western blotting, and the like. Several types of ELISA are commonly used, including direct ELISA, indirect ELISA and sandwich ELISA.

5.4. Test subject and sample

In certain embodiments, the various methods provided herein use a sample (e.g., a biological sample) from a subject or individual (e.g., a patient). The subject can be a patient, e.g., a patient having cancer (e.g., lymphoma, MM, or leukemia). The subject may be a mammal, e.g., a human. The subject may be male or female, and may be an adult, child or infant. The sample may be analyzed at a time within the active phase of the cancer (e.g., lymphoma, MM, or leukemia) or when the cancer (e.g., lymphoma, MM, or leukemia) is inactive. In certain embodiments, more than one sample may be obtained from one subject.

In certain embodiments, the sample used in the methods provided herein comprises a bodily fluid from a subject. Non-limiting examples of bodily fluids include blood (e.g., whole blood), plasma, amniotic fluid, aqueous humor, bile, cerumen, cowper's fluid, pre-ejaculatory fluid, chyle, chyme, female ejaculate, interstitial fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, tears, urine, vaginal lubrication, vomit, water, feces, internal bodily fluids (including cerebrospinal fluid around the brain and spinal cord), synovial fluid, intracellular fluid (fluid within cells), and vitreous fluid (fluid within the eye globe). In some embodiments, the sample is a blood sample. As edited in e.g. Innis et al may be used,PCR Protocolsobtaining a blood sample using conventional techniques described in (Academic Press,1990) leukocytes can be isolated from blood samples using conventional techniques or commercially available kits, such as the RosetteSep kit (Stein Cell Technologies, Vancouver, Canada). Subpopulations of leukocytes (e.g., monocytes, B cells, T cells, monocytes, granulocytes, or lymphocytes) can be further isolated using conventional techniques (e.g., Magnetic Activated Cell Sorting (MACS) (Miltenyi Biotec, Aubur, ca)) or Fluorescence Activated Cell Sorting (FACS) (Becton Dickinson, san jose, ca)).

In one embodiment, the blood sample is from about 0.1mL to about 10.0mL, from about 0.2mL to about 7mL, from about 0.3mL to about 5mL, from about 0.4mL to about 3.5mL, or from about 0.5mL to about 3 mL. In another embodiment, the blood sample is about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, or about 10.0 mL.

In some embodiments, the sample used in the methods of the invention comprises a biopsy (e.g., a tumor biopsy). The biopsy may be from any organ or tissue, such as skin, liver, lung, heart, colon, kidney, bone marrow, tooth, lymph node, hair, spleen, brain, breast, or other organ. Any biopsy technique known to those skilled in the art may be used to isolate a sample from a subject, for example, an open biopsy, a closed biopsy, a core biopsy, an open biopsy, an excisional biopsy, or a fine needle aspiration biopsy.

In one embodiment, the sample used in the methods provided herein is obtained from the subject prior to the subject receiving treatment for the disease or disorder. In another embodiment, the sample is obtained from the subject during the time the subject is being treated for the disease or disorder. In another embodiment, the sample is obtained from the subject after the subject is treated for the disease or disorder. In various embodiments, the treatment comprises administering a compound (e.g., a compound provided in section 5.5, below) to the subject.

5.1. Cell type

In certain embodiments, a sample used in the methods provided herein comprises a plurality of cells, such as cancer (e.g., lymphoma, MM, or leukemia) cells. Such cells may include any type of cell, such as stem cells, blood cells (e.g., Peripheral Blood Mononuclear Cells (PBMCs)), lymphocytes, B cells, T cells, monocytes, granulocytes, immune cells, or cancer cells.

B cells (B lymphocytes) include, for example, plasma B cells, memory B cells, B1 cells, B2 cells, marginal zone B cells, and follicular B cells. B cells may express immunoglobulins (antibodies) and B cell receptors.

A combination of commercially available antibodies (e.g., antibodies from Quest Diagnostic (San Juan Capistrano, california)) or Dako (denmark)) can be used to obtain a particular cell population.

In certain embodiments, the cells in the methods provided herein are PBMCs. In certain embodiments, the sample used in the methods provided herein is from a diseased tissue, e.g., from an individual having cancer (e.g., lymphoma, MM, or leukemia).

In certain embodiments, the cell lines are used as disease models for evaluating the effect of compounds, studying the mechanism of action, or establishing reference levels of biomarkers, etc. In some embodiments, the cells used in the methods provided herein are from a cancer (e.g., AML) cell line. In certain embodiments, the cell is from a lymphoma cell line. In other embodiments, the cell is from a MM cell line. In other embodiments, the cells are from a leukemia cell line. In some embodiments, the leukemia cell line is a CLL cell line. In other embodiments, the leukemia cell line is an ALL cell line. In yet other embodiments, the leukemia cell line is a CML cell line. In yet other embodiments, the leukemia cell line is an AML cell line. In one embodiment, the AML cell line is the KG-1 cell line. In another embodiment, the AML cell line is the KG-1a cell line. In yet another embodiment, the AML cell line is a KASUMI-1 cell line. In yet another embodiment, the AML cell line is the NB4 cell line. In one embodiment, the AML cell line is the MV-4-11 cell line. In another embodiment, the AML cell line is the MOLM-13 cell line. In yet another embodiment, the AML cell line is an HL-60 cell line. In yet another embodiment, the AML cell line is the U-937 cell line. In one embodiment, the AML cell line is the OCI-AML2 cell line. In another embodiment, the AML cell line is the OCI-AML3 cell line. In yet another embodiment, the AML cell line is an HNT-34 cell line. In yet another embodiment, the AML cell line is the ML-2 cell line. In one embodiment, the AML cell line is the AML-193 cell line. In another embodiment, the AML cell line is the F36-P cell line. In yet another embodiment, the AML cell line is a KASUMI-3 cell line. In yet another embodiment, the AML cell line is the MUTZ-8 cell line. In one embodiment, the AML cell line is a GDM-1 cell line. In another embodiment, the AML cell line is the SIG-M5 cell line. In yet another embodiment, the AML cell line is a TF-1 cell line. In yet another embodiment, the AML cell line is a Nomo-1 cell line. In one embodiment, the AML cell line is the UT-7 cell line. In another embodiment, the AML cell line is the THP-1 cell line.

In certain embodiments, the methods provided herein can be used to detect gene rearrangements in cells from a healthy individual. In certain embodiments, the number of cells used in the methods provided herein can range from a single cell to about 10 ⁹ And (4) cells. In some embodiments, the number of cells used in the methods provided herein is about 1x10 ⁴ About 5x10 ⁴ About 1x10 ⁵ About 5x10 ⁵ About 1x10 ⁶ About 5x10 ⁶ About 1x10 ⁷ About 5x10 ⁷ About 1x10 ⁸ About 5x10 ⁸ Or about 1x10 ⁹ 。

The number and type of cells collected from a subject can be monitored, for example, by measuring changes in cell surface markers using standard cell detection techniques such as flow cytometry, cell sorting, immunocytochemistry (e.g., staining with tissue-specific or cell marker-specific antibodies), Fluorescence Activated Cell Sorting (FACS), Magnetic Activated Cell Sorting (MACS), examining the morphology of cells using light or confocal microscopy, and/or measuring changes in gene expression using techniques well known in the art, such as PCR and gene expression profiling. These techniques can also be used to identify cells that are positive for one or more specific markers.

In certain embodiments, the cell subpopulation is used in the methods provided herein. Methods of sorting and isolating specific cell populations are well known in the art and may be based on cell size, morphology, or intracellular or extracellular markers. Such methods include, but are not limited to, flow cytometry, flow sorting, FACS, bead-based separations such as magnetic cell sorting, size-based separations (e.g., sieves, obstacle arrays, or filters), sorting in microfluidic devices, antibody-based separations, sedimentation, affinity adsorption, affinity extraction, density gradient centrifugation, laser capture microdissection, and the like. FACS is a well-known method for separating particles, including cells, based on their fluorescent properties (Kamarch, Methods enzymol.1987,151: 150-. Laser excitation of the fluorescent moieties in the individual particles will generate a small amount of charge, allowing electromagnetic separation of the positive and negative particles from the mixture. In one embodiment, the cell surface marker-specific antibody or ligand is labeled with a different fluorescent label. The cells are processed through a cell sorter, allowing the cells to be separated based on their binding capacity to the antibody used. FACS sorted particles can be deposited directly into individual wells of a 96-well or 384-well plate to facilitate isolation and cloning.

In one embodiment, RNA (e.g., mRNA) or protein is purified from the tumor and the levels of the gene set are measured by mRNA or protein expression analysis. In certain embodiments, the level of the gene set is measured by transcriptome profiling, qRT-PCR, microarray, high throughput sequencing, or other similar methods known in the art. In other embodiments, the level of the gene set is measured by ELISA, flow cytometry, immunofluorescence, or other similar methods known in the art.

5.5. Compound (I)

Compounds suitable for use in the methods and formulations provided herein are compound D: 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide having the structure:

or a stereoisomer or mixture of stereoisomers thereof, an isotopologue, a pharmaceutically acceptable salt, a tautomer, a solvate, a hydrate, a co-crystal, a clathrate, or a polymorph thereof. In certain embodiments, compound D is 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide.

Compound D may be prepared according to the methods described in the examples provided herein or as described in U.S. patent No. 9,499,514, the disclosure of which is incorporated herein by reference in its entirety. The compounds may also be synthesized based on the teachings herein according to other methods apparent to those skilled in the art.

In certain embodiments, compound D is a solid. In certain embodiments, compound D is a hydrate. In certain embodiments, compound D is solvated. In certain embodiments, compound D is anhydrous.

In certain embodiments, compound D is amorphous. In certain embodiments, compound D is crystalline. In certain embodiments, compound D is the crystalline form described in U.S. publication No. 2017-0197934, filed on day 6/1 of 2017, which is incorporated herein by reference in its entirety.

Solid forms of compound D can be prepared according to the methods described in the publication us publication No. 2017-0197934 filed on 6.1.2017. Solid forms can also be prepared according to other methods apparent to those skilled in the art.

In one embodiment, compound D is polymorphic form a, form B, form C, form D, form E, or an amorphous form of 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide. Polymorphs of 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide are briefly described herein. In certain embodiments, compound D has the polymorph as described in U.S. publication No. 2019/0030018, the disclosure of which is incorporated herein by reference in its entirety, and portions of which are described in more detail below.

Form A of 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide

In certain embodiments, the formulations provided herein are prepared from form a of compound D.

In one embodiment, form a is an anhydrous form of compound D. In another embodiment, form a of compound D is crystalline.

In certain embodiments, form a is obtained by crystallization from certain solvent systems, such as solvent systems comprising one or more of the following solvents: acetone and a solvent mixture of isopropanol and water at room temperature. In certain embodiments, form a is obtained as an intermediate solid form from a slurry in ethanol/water (1:1), acetone, or acetonitrile at elevated temperature (e.g., about 50 ℃).

In certain embodiments, form a is substantially crystalline as indicated by, for example, X-ray powder diffraction measurements. In one embodiment, form a of compound D has an X-ray powder diffraction pattern substantially as shown in figure 2 of U.S. publication No. 2019/0030018.

In one embodiment, form a of compound D has one or more characteristic X-ray powder diffraction peaks at 2 Θ angles of about 11.5, 15.6, 16.6, 17.2, 18.1, 19.0, 19.6, 21.1, 23.2, or 24.8 degrees 2 Θ, as depicted in figure 2 of U.S. publication No. 2019/0030018. In another embodiment, form a of compound D has one, two, three, or four characteristic X-ray powder diffraction peaks at 2 Θ angles of about 15.6, 16.6, 17.2, or 24.8 degrees 2 Θ. In another embodiment, form a of compound D has one, two, three, four, five, six, or seven characteristic X-ray powder diffraction peaks, as listed in table a. In another embodiment, form a of compound D has one, two, or three characteristic X-ray powder diffraction peaks, as listed in table a.

TABLE A

In one embodiment, form a of compound D has an SEM image as shown in figure 3 of U.S. publication No. 2019/0030018.

In one embodiment, the Thermogravimetric (TGA) thermal map of the crystalline form of compound D substantially corresponds to the representative TGA thermal map as depicted in figure 4 of U.S. publication No. 2019/0030018. In certain embodiments, no TGA weight loss is observed for form a.

In one embodiment, the DSC thermogram of crystalline form a of compound D substantially corresponds to the one depicted in figure 5 of U.S. publication No. 2019/0030018. In certain embodiments, form a is characterized by a DSC profile comprising a melting event wherein the onset temperature is 229 ℃ and the heat of fusion is 118J/g.

In certain embodiments, form a is characterized by dynamic vapor sorption analysis. A representative Dynamic Vapor Sorption (DVS) isotherm diagram is shown in fig. 6 of U.S. publication No. 2019/0030018. In certain embodiments, form a exhibits a water absorption of less than 1.5%, less than 1.2%, or about 1.2% w/w when the relative humidity ("RH") is increased from about 0% to about 90% RH. In certain embodiments, form a comprises less than 0.1% water, as determined in a coulomb Karl Fischer (KF) titrator equipped with an oven sample processor set at 225 ℃.

In certain embodiments, by ¹ No significant degradation of form a or residual solvent was observed by H NMR (see figure 7 of U.S. publication No. 2019/0030018).

In certain embodiments, form a of compound D is characterized by its stability profile upon compression. In certain embodiments, form a is stable, e.g., the XRPD pattern remains substantially unchanged and the diffraction peak is broader after about 1 minute of application of 2000-psi pressure (see fig. 8 of U.S. publication No. 2019/0030018).

In yet another embodiment, form a of compound D is substantially pure. In certain embodiments, form a of substantially pure compound D is substantially free of other solid forms, e.g., amorphous forms. In certain embodiments, the purity of substantially pure form a of compound D is not less than about 95% pure, not less than about 96% pure, not less than about 97% pure, not less than about 98% pure, not less than about 98.5% pure, not less than about 99% pure, not less than about 99.5% pure, or not less than about 99.8% pure.

In certain embodiments, form a of compound D is substantially pure. In certain embodiments herein, form a of compound D is substantially free of other solid forms comprising compound D, including, for example, form B, C, D, E, and/or amorphous solid forms comprising compound D. In certain embodiments, form a is a mixture comprising a solid form of compound D, including, for example, a mixture comprising one or more of: form B, C, D, E, and an amorphous solid form comprising compound D.

Form B of 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide

In certain embodiments, the formulations provided herein are prepared from amorphous form B of compound D.

In certain embodiments, form B is obtained by anti-solvent recrystallization from certain solvent systems, such as solvent systems comprising one or more of the following solvents: methanol/water, DMSO/isopropanol, DMSO/toluene, and DMSO/water. In certain embodiments, form B is obtained by cooling recrystallization from THF/water (1: 1).

In certain embodiments, form B is crystalline as indicated by, for example, X-ray powder diffraction measurements. In one embodiment, form B of compound D has an X-ray powder diffraction pattern substantially as shown in figure 9 of U.S. publication No. 2019/0030018.

In one embodiment, form B of compound D has one or more characteristic X-ray powder diffraction peaks at 2 Θ angles of about 15.4, 16.3, 16.7, 17.7, 20.4, 25.6, or 27.5 degrees 2 Θ, as depicted in figure 9 of U.S. publication No. 2019/0030018. In another embodiment, form B of compound D has one, two, three, or four characteristic X-ray powder diffraction peaks at 2 Θ angles of about 16.7, 25.6, 15.4, or 16.3 degrees 2 Θ. In another embodiment, form B of compound D has one, two, three, four, five, six, or seven characteristic X-ray powder diffraction peaks, as listed in table B. In another embodiment, form B of compound D has one, two, or three characteristic X-ray powder diffraction peaks, as listed in table B.

TABLE B

In one embodiment, form B of compound D has an SEM image as shown in figure 10 of U.S. publication No. 2019/0030018. In one embodiment, the Thermogravimetric (TGA) thermal map of the crystalline form of compound D substantially corresponds to the representative TGA thermal map as depicted in figure 11 of U.S. publication No. 2019/0030018. In certain embodiments, form B exhibits no TGA weight loss below 170 ℃. In certain embodiments, form B exhibits a TGA weight loss of 0.4% between 170 ℃ and 230 ℃.

In one embodiment, the DSC thermogram of crystalline form B of compound D substantially corresponds to the one depicted in figure 12 of U.S. publication No. 2019/0030018. In certain embodiments, form B is characterized by a DSC profile comprising a melting/recrystallization event at 219 ℃ -224 ℃ and a major melting event with a peak temperature of 231 ℃.

In certain embodiments, form B is characterized by a dynamic vapor sorption analysis. A representative Dynamic Vapor Sorption (DVS) isotherm diagram is shown in fig. 13 of U.S. publication No. 2019/0030018. In certain embodiments, form B exhibits a water uptake of about 1.4% w/w when the relative humidity ("RH") is increased from about 0% to about 90% RH. In certain embodiments, form B comprises less than 0.1% water, as determined in a coulomb Karl Fischer (KF) titrator equipped with an oven sample processor set at 225 ℃.

In some implementationsIn the scheme, through ¹ H NMR, form B, showed no significant degradation or residual solvent (see fig. 14 of U.S. publication No. 2019/0030018).

In certain embodiments, form B of compound D is characterized by its stability profile upon compression. In certain embodiments, form B is stable, e.g., the XRPD pattern remains substantially unchanged and the diffraction peak is broader after about 1 minute of 2000-psi application (see fig. 15 of U.S. publication No. 2019/0030018).

In yet another embodiment, form B of compound D is substantially pure. In certain embodiments, form B of substantially pure compound D is substantially free of other solid forms, e.g., amorphous forms. In certain embodiments, the purity of substantially pure form B of compound D is not less than about 95% pure, not less than about 96% pure, not less than about 97% pure, not less than about 98% pure, not less than about 98.5% pure, not less than about 99% pure, not less than about 99.5% pure, or not less than about 99.8% pure.

In certain embodiments, form B of compound D is substantially pure. In certain embodiments, form B of compound D is substantially free of other solid forms comprising compound D, including, for example, form A, C, D, E, and/or amorphous solid forms comprising compound D. In certain embodiments, form B is a mixture comprising a solid form of compound D, including, for example, a mixture comprising one or more of: form A, C, D, E, and an amorphous solid form comprising compound D.

Form C of 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide

In certain embodiments, the formulations provided herein are prepared from anhydrous form C of compound D. In certain embodiments, form C is the most thermodynamically stable anhydrate in the crystalline form of compound D.

In certain embodiments, form C is obtained by slurrying compound D in certain solvent systems, such as solvent systems comprising one or more of the following solvents, for an extended period of time: acetonitrile/water, acetone, or ethanol/water.

In certain aspects, form C is obtained by: form B (1X wt) is slurried in acetone (30X vol) at elevated temperature, e.g., from 60 ℃ to 80 ℃ or 70 ℃ to 75 ℃, for at least 24 hours, and the mixture is cooled to room temperature. In one aspect, the slurry is carried out at a temperature of 70 ℃ to 75 ℃ under a nitrogen pressure of 50 to 55 psi. In one aspect, the mixture is cooled to room temperature over at least 6 hours.

In certain embodiments, form C is crystalline as indicated by, for example, X-ray powder diffraction measurements. In one embodiment, form C of compound D has an X-ray powder diffraction pattern substantially as shown in figure 16 of U.S. publication No. 2019/0030018.

In one embodiment, form C of compound D has one or more characteristic X-ray powder diffraction peaks at 2 Θ angles of about 7.4, 11.5, 15.8, 16.7, 16.9, 17.7, 18.4, 19.2, 19.5, 21.1, 23.4, 24.7, or 29.9 degrees 2 Θ, as depicted in figure 16 of U.S. publication No. 2019/0030018. In another embodiment, form C of compound D has one, two, three, or four characteristic X-ray powder diffraction peaks at 2-theta angles of about 16.7, 16.9, 17.7, or 24.7 degrees 2 theta. In another embodiment, form C of compound D has one, two, three, four, five, six, or seven characteristic X-ray powder diffraction peaks, as listed in table C. In another embodiment, form C of compound D has one, two, or three characteristic X-ray powder diffraction peaks, as listed in table C.

Watch C

In one embodiment, form C of compound D has the SEM image shown in figure 17 of U.S. publication No. 2019/0030018. In one embodiment, the Thermogravimetric (TGA) thermal map of the crystalline form of compound D substantially corresponds to the representative TGA thermal map as depicted in figure 18 of U.S. publication No. 2019/0030018. In certain embodiments, form C exhibits no TGA weight loss.

In one embodiment, the DSC thermogram of the crystalline form C of compound D substantially corresponds to the one depicted in figure 19 of U.S. publication No. 2019/0030018. In certain embodiments, form C is characterized by a DSC profile comprising a melting event, wherein the onset temperature is 232 ℃ and the heat of fusion is 126J/g.

In certain embodiments, form C is characterized by dynamic vapor sorption analysis. A representative Dynamic Vapor Sorption (DVS) isotherm diagram is shown in fig. 20 of U.S. publication No. 2019/0030018. In certain embodiments, form C exhibits a water uptake of about 0.6% w/w when the relative humidity ("RH") is increased from about 0% to about 90% RH. In certain embodiments, form C comprises less than 0.1% water, as determined in a coulomb Karl Fischer (KF) titrator equipped with an oven sample processor set at 225 ℃.

In certain embodiments, by ¹ H NMR, form C, showed no significant degradation or residual solvent (see fig. 21 of U.S. publication No. 2019/0030018).

In certain embodiments, form C of compound D is characterized by its stability profile upon compression. In certain embodiments, form C is stable, e.g., the XRPD pattern remains substantially unchanged and the diffraction peak is broader after about 1 minute of 2000-psi application (see fig. 22 of U.S. publication No. 2019/0030018).

In yet another embodiment, form C of compound D is substantially pure. In certain embodiments, form C of substantially pure compound D is substantially free of other solid forms, e.g., amorphous forms. In certain embodiments, the purity of substantially pure form C of compound D is not less than about 95% pure, not less than about 96% pure, not less than about 97% pure, not less than about 98% pure, not less than about 98.5% pure, not less than about 99% pure, not less than about 99.5% pure, or not less than about 99.8% pure.

In certain embodiments, form C of compound D is substantially pure. In certain embodiments, form C of compound D is substantially free of other solid forms comprising compound D, including, for example, form A, B, D, E, and/or amorphous solid forms comprising compound D. In certain embodiments, form C is a mixture comprising a solid form of compound D, including, for example, a mixture comprising one or more of: form A, B, D, E, and an amorphous solid form comprising compound D.

Form D of 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide

In certain embodiments, the formulations provided herein are prepared from form D of compound D. In certain embodiments, form D of compound D is a DMSO solvate.

In certain embodiments, form D is obtained by heating form B in DMSO/methyl isobutyl ketone and cooling the solution.

In certain embodiments, form D is crystalline as indicated by, for example, X-ray powder diffraction measurements. In one embodiment, form D of compound D has an X-ray powder diffraction pattern substantially as shown in figure 23 of U.S. publication No. 2019/0030018.

In one embodiment, form D of compound D has one or more characteristic X-ray powder diffraction peaks at 2 Θ angles of about 14.1, 14.3, 18.8, 19.1, 23.6, or 24.0 degrees 2 Θ, as depicted in figure 23 of U.S. publication No. 2019/0030018. In another embodiment, form D of compound D has one, two, three, or four characteristic X-ray powder diffraction peaks at 2-theta angles of about 14.1, 14.3, 18.8, or 19.1 degrees 2 theta. In another embodiment, form D of compound D has one, two, three, four, five, six, or seven characteristic X-ray powder diffraction peaks, as set forth in table D. In another embodiment, form D of compound D has one, two, or three characteristic X-ray powder diffraction peaks, as listed in table D.

Watch D

In one embodiment, provided herein is a crystalline form of compound D having a Thermogravimetric (TGA) thermal map substantially corresponding to the representative TGA thermal map as depicted in figure 24 of U.S. publication No. 2019/0030018. In certain embodiments, form D exhibits a TGA weight loss of up to about 14.1% at 140 ℃.

In certain embodiments, form D comprises about 14.3 wt% DMSO as measured by gas chromatography.

In yet another embodiment, form D of compound D is substantially pure. In certain embodiments, form D of substantially pure compound D is substantially free of other solid forms, e.g., amorphous forms. In certain embodiments, the purity of substantially pure form D of compound D is not less than about 95% pure, not less than about 96% pure, not less than about 97% pure, not less than about 98% pure, not less than about 98.5% pure, not less than about 99% pure, not less than about 99.5% pure, or not less than about 99.8% pure.

In certain embodiments, form D of compound D is substantially pure. In certain embodiments, form D of compound D is substantially free of other solid forms comprising compound D, including, for example, form A, B, C, E, and/or amorphous solid forms comprising compound D as provided herein. In certain embodiments, form D is a mixture comprising a solid form of compound D, including, for example, a mixture comprising one or more of: form A, B, C, E, and an amorphous solid form comprising compound D.

Form E of 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide

In certain embodiments, the formulations provided herein are prepared from form E of compound D. In certain embodiments, form E of compound D is a DMSO solvate.

In certain embodiments, form E is obtained from form C in DMSO/MIBK or DMSO/IPA or DMSO/anisole at room temperature.

In certain embodiments, form E is crystalline as indicated by, for example, X-ray powder diffraction measurements. In one embodiment, form E of compound D has an X-ray powder diffraction pattern substantially as shown in figure 25 of U.S. publication No. 2019/0030018.

In one embodiment, form E of compound D has one or more characteristic X-ray powder diffraction peaks at 2-theta angles of about 10.5, 12.5, 16.1, 17.0, 18.5, 21.2, 21.7, 22.6, 22.9, 23.4, 23.8, 24.1, 25.1, or 26.7 degrees 2 theta as depicted in figure 25 of U.S. publication No. 2019/0030018. In another embodiment, form E of compound D has one, two, three, or four characteristic X-ray powder diffraction peaks at 2-theta angles of about 16.1, 17.0, 21.2, or 22.9 degrees 2 theta. In another embodiment, form E of compound D has one, two, three, four, five, six, or seven characteristic X-ray powder diffraction peaks, as set forth in table E. In another embodiment, form E of compound D has one, two, or three characteristic X-ray powder diffraction peaks, as listed in table E.

TABLE E

In one embodiment, provided herein is a crystalline form of compound D having a Thermogravimetric (TGA) thermal map substantially corresponding to the representative TGA thermal map as depicted in figure 26 of U.S. publication No. 2019/0030018. In certain embodiments, form E exhibits a TGA weight loss of about 19.4% up to 120 ℃. In certain embodiments, form E exhibits an additional weight loss of 24.9% between 120 ℃ and 220 ℃.

In one embodiment, form E of compound D is substantially pure. In certain embodiments, form E of substantially pure compound D is substantially free of other solid forms, e.g., amorphous forms. In certain embodiments, form E of substantially pure compound D is not less than about 95% pure, not less than about 96% pure, not less than about 97% pure, not less than about 98% pure, not less than about 98.5% pure, not less than about 99% pure, not less than about 99.5% pure, or not less than about 99.8% pure.

In certain embodiments, form E of compound D is substantially pure. In certain embodiments herein, form E of compound D is substantially free of other solid forms comprising compound D, including, for example, form A, B, C, D, and/or amorphous solid forms comprising compound D. In certain embodiments, form E is a mixture comprising a solid form of compound D, including, for example, a mixture comprising one or more of: form A, B, C, D, and an amorphous solid form comprising compound D.

Amorphous forms of 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide

In certain embodiments, the formulations provided herein comprise amorphous compound D.

In certain embodiments, provided herein are methods of making an amorphous form by heating compound D in THF and water and cooling the solution.

In one embodiment, provided herein is an amorphous solid form of compound D having a modulated DSC thermogram as depicted in figure 27 of U.S. publication No. 2019/0030018.

In one embodiment, amorphous compound D has an X-ray powder diffraction pattern substantially as shown in figure 28 of U.S. publication No. 2019/0030018.

In one embodiment, amorphous compound D has the structure substantially as shown in figure 29 of U.S. publication No. 2019/0030018 ¹ H NMR spectrum。

In yet another embodiment, amorphous compound D is substantially pure. In certain embodiments, substantially pure amorphous compound D is substantially free of other solid forms, such as form a, form B, form C, form D, or form E. In certain embodiments, the purity of substantially pure amorphous compound D is not less than about 95% pure, not less than about 96% pure, not less than about 97% pure, not less than about 98% pure, not less than about 98.5% pure, not less than about 99% pure, not less than about 99.5% pure, or not less than about 99.8% pure.

Isotopologues of 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide

Also provided herein are isotopically enriched analogs ("isotopologues") of 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide provided herein. Isotopic enrichment (e.g., deuteration) of drugs to improve pharmacokinetics ("PK"), pharmacodynamics ("PD"), and toxicity profiles have previously been demonstrated with certain classes of drugs. See, e.g., lijin nsky et al, Food cosmet. toxicol, 20:393 (1982); lijin insky et al, j.nat. cancer inst.,69:1127 (1982); mangold et al, Mutation Res.308:33 (1994); gordon et al, Drug meta. dispos, 15:589 (1987); zello et al, Metabolism,43:487 (1994); gately et al, J.Nucl.Med.,27:388 (1986); wade D, chem.biol.interact.117:191 (1999).

Without being bound by any particular theory, isotopic enrichment of a drug can be used, for example, (1) to reduce or eliminate unwanted metabolites, (2) to increase the half-life of the parent drug, (3) to reduce the dose required to achieve a desired effect, (4) to reduce the dose required to achieve a desired effect, (5) to increase the formation of active metabolites, if formed, and/or (6) to reduce the production of harmful metabolites in specific tissues and/or to create more potent drugs and/or safer drugs for combination therapy, whether or not the combination therapy is intended.

Substitution of an atom for one of its isotopes will generally result in a change in the reaction rate of the chemical reaction. This phenomenon is known as the kinetic isotope effect ("KIE"). For example, if a C-H bond is broken in the rate-limiting step (i.e., the step with the highest transition state energy) in a chemical reaction, substitution of that hydrogen with deuterium will result in a decrease in the reaction rate and the process will slow down. This phenomenon is known as the deuterium kinetic isotope effect ("DKIE"). (see, e.g., Foster et al, adv. drug Res., Vol.14, pp.1-36 (1985); Kushner et al, Can. J. Physiol. Pharmacol., Vol.77, pp.79-88 (1999)).

The size of DKIE can be expressed as the ratio between the rate of a given reaction in which the C — H bond is broken and the rate of the same reaction in which deuterium replaces hydrogen. DKIE can range from about 1 (no isotopic effect) to very large numbers, such as 50 or more, meaning that the reaction can be fifty or more times slower when deuterium is substituted for hydrogen. Without being bound by a particular theory, the high DKIE value may be due in part to a phenomenon known as tunneling, which is a result of uncertain principles. Tunneling is due to the small mass of the hydrogen atoms and occurs because transition states involving protons can sometimes form in the absence of the required activation energy. Since deuterium is of a greater mass than hydrogen, it is statistically much less likely to occur.

Tritium ("T") is a radioactive isotope of hydrogen used in research, fusion reactors, neutron generators, and radiopharmaceuticals. Tritium is a hydrogen atom with 2 neutrons in the nucleus and an atomic weight close to 3. It occurs naturally in the environment at very low concentrations, most often as T ₂ O is present. Tritium decays slowly (half-life-12.3 years) and releases low energy beta particles that cannot penetrate the outer layers of human skin. Internal exposure is a major hazard associated with this isotope, but it must be ingested in large quantities to pose a significant health risk. Compared to deuterium, a smaller amount of tritium must be consumed before it reaches dangerous levels. Replacement of hydrogen with tritium ("T") results in a stronger bond than deuterium and produces a numerically greater isotopic effect.

Similarly, other elements are replaced with isotopes (including but not limited to ¹³ C or ¹⁴ C is substituted carbon, and the reaction is carried out, ³³ S、 ³⁴ s or ³⁶ S is substituted for sulfur, and the sulfur is replaced by sulfur, ¹⁵ n is substituted with nitrogen, and ¹⁷ o or ¹⁸ O instead of oxygen) will provide similar kinetic isotope effects.

In certain embodiments, a compound provided herein is a prodrug of a compound provided herein (e.g., a prodrug of compound D). Exemplary compounds include those disclosed in U.S. publication No. 2017/0197933, the disclosure of which is incorporated herein by reference in its entirety.

5.6. Pharmaceutical composition

In some embodiments, the compounds provided herein are formulated as pharmaceutical compositions. In some embodiments, compound D is provided as a stable formulation of compound D. In one embodiment, the formulation of compound D comprises a solid form of 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide. In one embodiment, the formulation of compound D comprises an amorphous form of 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide.

In certain embodiments, the formulations are prepared using dimethyl sulfoxide as a co-solvent or processing aid. In certain embodiments, the formulations are prepared using formic acid as a co-solvent or processing aid. In certain embodiments, the formulations are prepared without any co-solvents or processing aids.

In certain embodiments, the formulation comprises dimethyl sulfoxide as a co-solvent or processing aid. In certain embodiments, the formulation comprises formic acid as a co-solvent or processing aid. In certain embodiments, the formulation does not contain any co-solvents or processing aids.

In certain embodiments, the formulations provided herein are lyophilized formulations. In certain embodiments, the formulations provided herein are reconstituted formulations obtained in a pharmaceutically acceptable solvent to produce a pharmaceutically acceptable solution.

Formulation Ia

In one embodiment, provided herein are formulations comprising compound D in an amount of about 0.05-0.2%, citrate buffer in an amount of about 3-6%, and hydroxypropyl β -cyclodextrin (HPBCD) in an amount of about 92-98%, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.05% -0.2%, citrate buffer in an amount of about 3% -6%, and sulfobutyl ether- β -cyclodextrin in an amount of about 92% -98%, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.05-0.2%, citrate buffer in an amount of about 3-6%, HPBCD in an amount of about 92-98%, and no more than about 1% dimethylsulfoxide, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.05% -0.2%, citrate buffer in an amount of about 3% -6%, sulfobutyl ether- β -cyclodextrin in an amount of about 92% -98%, and no more than about 1% dimethyl sulfoxide, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.08% -0.15%, citrate buffer in an amount of about 3% -6%, and HPBCD in an amount of about 94% -96%, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.08% -0.15%, citrate buffer in an amount of about 3% -6%, and sulfobutyl ether- β -cyclodextrin in an amount of about 94% -96%, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.08% -0.15%, citrate buffer in an amount of about 3% -6%, HPBCD in an amount of about 94% -96%, and no more than about 1% dimethylsulfoxide, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.08% -0.15%, citrate buffer in an amount of about 3% -6%, sulfobutyl ether- β -cyclodextrin in an amount of about 94% -96%, and no more than about 1% dimethyl sulfoxide, based on the total weight of the formulation.

In one aspect, the formulations provided herein comprise compound D in an amount of about 0.08% to about 0.15% based on the total weight of the formulation. In certain embodiments, the amount of compound D is about 0.09% to about 0.15%, about 0.1% to about 0.13%, or about 0.11% to about 0.12%, based on the total weight of the formulation. In certain embodiments, the amount of compound D is about 0.05%, 0.07%, 0.09%, 0.11%, 0.12%, 0.13%, or 0.15% based on the total weight of the formulation. In one embodiment, the amount of compound D in the formulation is about 0.12% based on the total weight of the formulation.

In another aspect, provided herein are formulations comprising compound D in an amount of about 0.5mg to about 2mg in a 20-cc vial. Yet another aspect is a formulation comprising compound D in an amount of about 0.5mg to about 1.5mg, about 0.75mg to about 1.25mg, or about 0.8mg to about 1.1mg in a 20-cc vial. In one aspect, compound D is present in an amount of about 0.7, 0.75, 0.76, 0.8, 0.9, 1.0, 1.05, or 1.2mg in a 20-cc vial. In one aspect, compound D is present in an amount of about 1.05mg in a 20-cc vial.

In one aspect, the formulations provided herein contain citrate buffer. In one aspect, the amount of citrate buffer in the formulations provided herein is from about 3% to about 6% based on the total weight of the formulation. In one aspect, the amount of citrate buffer in a formulation provided herein is about 3%, 3.5%, 4%, 4.2%, 4.5%, or 5% based on the total weight of the formulation. In one aspect, the amount of citrate buffer in the formulations provided herein is about 4.2% based on the total weight of the formulation. In one aspect, the amount of citrate buffer in a formulation provided herein is about 37mg in a 20cc vial.

In one embodiment, the citrate buffer includes anhydrous citric acid and anhydrous sodium citrate. In certain embodiments, the amount of anhydrous citric acid is from about 1.5% to about 3%, from about 1.75% to about 2.75%, or from about 2% to about 2.5%, based on the total weight of the formulation. In certain embodiments, the amount of anhydrous citric acid in the formulation is about 1.5%, 1.75%, 2%, 2.1%, or 2.5% based on the total weight of the formulation. In one embodiment, the amount of anhydrous citric acid in the formulation is about 2%, 2.1%, 2.22%, or 2.3% based on the total weight of the formulation. In one embodiment, the amount of anhydrous citric acid in the formulation is about 2.10% based on the total weight of the formulation.

Yet another aspect is a formulation comprising anhydrous citric acid in an amount of from about 16mg to about 20mg in a 20-cc vial. In one embodiment, the amount of anhydrous citric acid is about 16, 17, 18, 18.2, 18.4, 18.6, 18.8, 19, or 20mg in a 20-cc vial. In one embodiment, the amount of anhydrous citric acid is about 18.6mg in a 20-cc vial.

In certain embodiments, the amount of anhydrous sodium citrate is from about 1.5% to about 3%, from about 1.75% to about 2.75%, or from about 2% to about 2.5%, based on the total weight of the formulation. In certain embodiments, the amount of anhydrous sodium citrate in the formulation is about 1.5%, 1.75%, 2%, 2.1%, or 2.5% based on the total weight of the formulation. In one embodiment, the amount of anhydrous sodium citrate in the formulation is about 2%, 2.05%, 2.08%, or 2.1% based on the total weight of the formulation. In one embodiment, the amount of anhydrous sodium citrate in the formulation is about 2.08% based on the total weight of the formulation.

Yet another aspect is a formulation comprising anhydrous sodium citrate in an amount of about 16mg to about 20mg in a 20-cc vial. In one embodiment, the amount of anhydrous sodium citrate is about 16, 17, 18, 18.2, 18.4, 18.6, 18.8, 19, or 20mg in a 20-cc vial. In one embodiment, the amount of anhydrous sodium citrate is about 18.4mg in a 20-cc vial.

In certain embodiments, the amount of HPBCD in the formulations provided herein is about 94% to about 97%, based on the total weight of the formulation. In one embodiment, the amount of HPBCD in the formulations provided herein is about 94.5%, 95%, 95.5%, or 96% based on the total weight of the formulation. In one embodiment, the amount of HPBCD in the formulations provided herein is about 95% based on the total weight of the formulation.

In certain embodiments, the amount of sulfobutyl ether- β -cyclodextrin in the formulations provided herein is from about 94% to about 97%, based on the total weight of the formulation. In one embodiment, the amount of sulfobutyl ether- β -cyclodextrin in the formulations provided herein is about 94.5%, 95%, 95.5%, or 96% based on the total weight of the formulation. In one embodiment, the amount of sulfobutyl ether- β -cyclodextrin in the formulations provided herein is about 95% based on the total weight of the formulation.

Another aspect is a formulation comprising HPBCD in an amount of about 800 to 900mg in a 20-cc vial. Another aspect is a formulation comprising HPBCD in an amount of about 810 to 880mg, 820 to 860mg, or 830 to 850mg in a 20-cc vial. Another aspect is a formulation comprising HPBCD in an amount of about 840mg in a 20-cc vial.

Another aspect is a formulation comprising sulfobutyl ether- β -cyclodextrin in an amount of about 800 to 900mg in a 20-cc vial. Another aspect is a formulation comprising sulfobutyl ether- β -cyclodextrin in an amount of about 810 to 880mg, 820 to 860mg, or 830 to 850mg in a 20-cc vial. Another aspect is a formulation comprising sulfobutyl ether- β -cyclodextrin in an amount of about 840mg in a 20-cc vial.

Another aspect is an amount of about 840mg contained in a 20-cc vial

Formulation of HPB.

In one embodiment, the formulation comprises dimethyl sulfoxide in an amount of no more than about 1.5% based on the total weight of the formulation. In one embodiment, the formulation comprises dimethyl sulfoxide in an amount up to 0.1%, 0.2%, 0.3%, 0.4%, 0.6%, 0.7%, 0.8%, 0.9% or 1% based on the total weight of the formulation. In one embodiment, the formulation comprises no more than about 0.1%, 0.2%, 0.3%, 0.4%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% dimethyl sulfoxide, based on the total weight of the formulation. In one embodiment, the formulation comprises dimethyl sulfoxide in an amount up to about 0.1% to about 1.5% based on the total weight of the formulation. In one embodiment, the amount of dimethyl sulfoxide in the formulations provided herein is from about 0.1% to about 1.3% based on the total weight of the formulation. In one embodiment, the amount of dimethyl sulfoxide in the formulations provided herein is about 0.1%, 0.2%, 0.3%, 0.4%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% based on the total weight of the formulation. In one embodiment, the formulations provided herein do not contain any dimethyl sulfoxide. In one embodiment, the amount of dimethyl sulfoxide in the formulations provided herein is about 0.4% to 0.8% based on the total weight of the formulation.

Another aspect is a formulation comprising dimethyl sulfoxide in an amount of about 4 to 7mg in a 20-cc vial. Another aspect is a formulation comprising dimethyl sulfoxide in an amount of about 4.5 to 6.5mg or 5 to 6mg in a 20-cc vial.

In certain embodiments, the formulations provided herein are lyophilized, and the lyophilized formulations upon reconstitution have a pH of about 4 to 5. In certain embodiments, the reconstituted formulation has a pH of about 4.2 to 4.4. In one embodiment, the lyophilized formulation after reconstitution has a pH of about 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.

In certain embodiments, the lyophilized formulation after reconstitution has an osmolality of about 250-290 mOsm/kg. In certain embodiments, the lyophilized formulation after reconstitution has an osmolality of about 260 and 280 mOsm/kg.

In certain embodiments, provided herein are containers comprising the formulations provided herein. In one aspect, the container is a glass vial. In one aspect, the container is a 20-cc glass vial.

In one aspect, provided herein is a formulation in a 20-cc vial, the formulation comprising: compound D in an amount to provide 1.05mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; and a pharmaceutically acceptable carrier or excipient comprising a bulking agent as described herein. In one embodiment, the formulation further comprises no more than about 7mg of dimethyl sulfoxide as a residual solvent. In one embodiment, the formulation comprises no more than about 6mg of dimethyl sulfoxide as a residual solvent. In one embodiment, the formulation comprises no more than about 5mg of dimethyl sulfoxide as a residual solvent. In one embodiment, the formulation comprises no more than about 4mg of dimethyl sulfoxide as a residual solvent. In one embodiment, the formulation comprises about 3mg to about 7mg, about 4mg to about 6mg, about 4mg to about 5mg, or about 5mg to about 6mg of dimethyl sulfoxide as a residual solvent. In one embodiment, the formulation comprises about 4, 4.5, 5, 5.3, 5.5, 5.7, 6, or 6.5mg of dimethyl sulfoxide as residual solvent.

In one embodiment, provided herein is a formulation consisting essentially of: compound D in an amount of about 0.05% -0.2%, citrate buffer in an amount of about 3% -6%, and HPBCD in an amount of about 92% -98%, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation consisting essentially of: compound D in an amount of about 0.05% -0.2%, citrate buffer in an amount of about 3% -6%, and sulfobutyl ether- β -cyclodextrin in an amount of about 92% -98%, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation consisting essentially of: compound D in an amount of about 0.05% -0.2%, citrate buffer in an amount of about 3% -6%, HPBCD in an amount of about 92% -98%, and no more than about 1% dimethyl sulfoxide, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation consisting essentially of: compound D in an amount of about 0.05% -0.2%, citrate buffer in an amount of about 3% -6%, sulfobutyl ether- β -cyclodextrin in an amount of about 92% -98%, and no more than about 1% dimethyl sulfoxide, based on the total weight of the formulation.

In one aspect, provided herein is a formulation in a 20-cc vial, the formulation comprising: compound D in an amount to provide 1.05mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; a pharmaceutically acceptable carrier or excipient comprising a buffer and bulking agent as described herein; and about 5mg to about 6mg of dimethyl sulfoxide as a residual solvent. Buffers and extenders may be present in amounts as described herein.

In one aspect, provided herein is a formulation in a 20-cc vial, the formulation comprising: compound D in an amount to provide 1.05mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 18.6mg of anhydrous citric acid; 18.4mg of anhydrous sodium citrate; 840mg HPBCD; and about 5mg to about 6mg of dimethyl sulfoxide as a residual solvent, as described herein. In one embodiment, the formulation in a 20-cc vial is reconstituted with 3.8mL sterile water for injection.

In one aspect, provided herein is a formulation in a 20-cc vial consisting essentially of: compound D in an amount to provide 1.05mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 18.6mg of anhydrous citric acid; 18.4mg of anhydrous sodium citrate; 840mg HPBCD; and about 5mg to about 6mg of dimethyl sulfoxide as a residual solvent, as described herein. In one embodiment, the formulation in a 20-cc vial is reconstituted with 3.8mL sterile water for injection.

In one aspect, provided herein is a formulation in a 20-cc vial, the formulation consisting of: compound D in an amount to provide 1.05mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 18.6mg of anhydrous citric acid; 18.4mg of anhydrous sodium citrate; 840mg HPBCD; and about 5mg to about 6mg of dimethyl sulfoxide as a residual solvent, as described herein. In one embodiment, the formulation in a 20-cc vial is reconstituted with 3.8mL sterile water for injection.

In one embodiment, provided herein is an aqueous formulation comprising compound D in an amount of about 0.05-0.2% based on the total weight of solids, citrate buffer in an amount of about 3-6% based on the total weight of solids, HPBCD in an amount of about 92-98% based on the total weight of solids, and a diluent.

In one embodiment, provided herein is an aqueous formulation consisting essentially of: compound D in an amount of about 0.05% -0.2% based on the total weight of solids, citrate buffer in an amount of about 3% -6% based on the total weight of solids, HPBCD in an amount of about 92% -98% based on the total weight of solids, and a diluent.

In one aspect, provided herein is an aqueous formulation comprising: compound D in an amount to provide 1.05mg2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 18.6mg of anhydrous citric acid; 18.4mg of anhydrous sodium citrate; 840mg HPBCD; and about 5mg to about 6mg of dimethyl sulfoxide as a residual solvent and about 3.8mL of diluent.

In one aspect, provided herein is an aqueous formulation consisting essentially of: compound D in an amount to provide 1.05mg2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 18.6mg of anhydrous citric acid; 18.4mg of anhydrous sodium citrate; 840mg HPBCD; and about 5mg to about 6mg of dimethyl sulfoxide as a residual solvent and about 3.8mL of diluent.

In one aspect, provided herein is an aqueous formulation consisting of: compound D in an amount to provide 1.05mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 18.6mg of anhydrous citric acid; 18.4mg of anhydrous sodium citrate; 840mg HPBCD; and about 5mg to about 6mg of dimethyl sulfoxide as a residual solvent and about 3.8mL of diluent.

Formulation Ib

In one embodiment, provided herein are formulations comprising compound D in an amount of about 0.01-0.15%, hydroxypropyl β -cyclodextrin in an amount of about 99.1-99.99%. In one embodiment, provided herein are formulations comprising compound D in an amount of about 0.01-0.15%, hydroxypropyl β -cyclodextrin in an amount of about 99.1-99.99%, and no more than about 0.5% formic acid, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.05-0.25% and HPBCD in an amount of about 99.1-99.9% based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.05-0.25%, HPBCD in an amount of about 99.1-99.9%, and no more than about 0.5% formic acid, based on the total weight of the formulation.

In one embodiment, provided herein are formulations comprising compound D in an amount of about 0.05-0.25% and HPBCD in an amount of about 99.75-99.9% based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.05-0.25%, HPBCD in an amount of about 99.75-99.9%, and no more than about 0.5% formic acid, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.05-0.25%, HPBCD in an amount of about 99.75-99.9%, and no more than about 0.2% formic acid, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.08-0.15% and HPBCD in an amount of about 99.8-99.9% based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.08% -0.15%, HPBCD in an amount of about 99.8% -99.9%, and no more than about 0.5% formic acid, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.08% -0.15%, HPBCD in an amount of about 99.8% -99.9%, and no more than about 0.12% formic acid, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.12% and HPBCD in an amount of about 99.88% based on the total weight of the formulation.

In one embodiment, provided herein are formulations comprising compound D in an amount of about 0.05-0.25% and sulfobutyl ether- β -cyclodextrin in an amount of about 99.1-99.9% based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.05-0.25%, sulfobutyl ether- β -cyclodextrin in an amount of about 99.1-99.9%, and no more than about 0.5% formic acid, based on the total weight of the formulation.

In one embodiment, provided herein are formulations comprising compound D in an amount of about 0.05-0.25% and sulfobutyl ether- β -cyclodextrin in an amount of about 99.75-99.9% based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.08% -0.15% and sulfobutyl ether- β -cyclodextrin in an amount of about 99.8% -99.9% based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.08% -0.15%, sulfobutyl ether- β -cyclodextrin in an amount of about 99.8% -99.9%, and no more than about 0.5% formic acid, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.12% and sulfobutyl ether- β -cyclodextrin in an amount of about 99.88%, based on the total weight of the formulation.

In one aspect, the formulations provided herein comprise compound D in an amount of about 0.08% to about 0.15% based on the total weight of the formulation. In certain embodiments, the amount of compound D is about 0.09% to about 0.15%, about 0.1% to about 0.13%, or about 0.11% to about 0.12%, based on the total weight of the formulation. In certain embodiments, the amount of compound D is about 0.05%, 0.07%, 0.09%, 0.11%, 0.12%, 0.13%, or 0.15% based on the total weight of the formulation. In one embodiment, the amount of compound D in the formulation is about 0.12%, based on the total weight of the formulation.

In another aspect, provided herein are formulations comprising compound D in an amount of about 0.5mg to about 2mg in a 20-cc vial. Yet another aspect is a formulation comprising compound D in an amount of about 0.5mg to about 1.5mg, about 0.75mg to about 1.25mg, or about 0.8mg to about 1.1mg in a 20-cc vial. In one aspect, compound D is present in an amount of about 0.7, 0.75, 0.76, 0.8, 0.9, 1.0, 1.05, or 1.2mg in a 20-cc vial. In one aspect, compound D is present in an amount of about 1mg in a 20-cc vial.

In one embodiment, the amount of HPBCD in the formulations provided herein is about 97% to about 99.9% based on the total weight of the formulation. In one embodiment, the amount of HPBCD in the formulations provided herein is about 98% to about 99.9% based on the total weight of the formulation. In one embodiment, the amount of HPBCD in the formulations provided herein is about 99.1%, 99.3%, 99.5%, 99.7%, or 99.9% based on the total weight of the formulation. In one embodiment, the amount of HPBCD in the formulations provided herein is about 99.5% based on the total weight of the formulation. Another aspect is a formulation comprising HPBCD in an amount of about 750-850mg in a 20-cc vial. Another aspect is a formulation comprising HPBCD in an amount of about 790 to 840mg, 780 to 830mg, or 790 to 810mg in a 20-cc vial. Another aspect is a formulation comprising HPBCD in an amount of about 800mg in a 20-cc vial.

Another aspect is a formulation comprising Kleptose HPB in an amount of about 800mg in a 20-cc vial.

In one embodiment, the amount of sulfobutyl ether- β -cyclodextrin in the formulations provided herein is from about 97% to about 99.9% based on the total weight of the formulation. In one embodiment, the amount of sulfobutyl ether- β -cyclodextrin in the formulations provided herein is from about 98% to about 99.9% based on the total weight of the formulation. In one embodiment, the amount of sulfobutyl ether- β -cyclodextrin in the formulations provided herein is about 99.1%, 99.3%, 99.5%, 99.7%, or 99.9% based on the total weight of the formulation. In one embodiment, the amount of sulfobutyl ether- β -cyclodextrin in the formulations provided herein is about 99.5% based on the total weight of the formulation.

Another aspect is a formulation comprising sulfobutyl ether- β -cyclodextrin in an amount of about 750 to 850mg in a 20-cc vial. Another aspect is a formulation comprising sulfobutyl ether- β -cyclodextrin in an amount of about 790 to 840mg, 780 to 830mg, or 790 to 810mg in a 20-cc vial. Another aspect is a formulation comprising sulfobutyl ether- β -cyclodextrin in an amount of about 800mg in a 20-cc vial.

In one embodiment, the formulation comprises no more than about 0.5% formic acid, based on the total weight of the formulation. In one embodiment, the formulation comprises formic acid in an amount up to about 0.05%, 0.07%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5% based on the total weight of the formulation. In one embodiment, the formulation comprises no more than about 0.05%, 0.07%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5% formic acid, based on the total weight of the formulation. In one embodiment, the amount of formic acid in the formulations provided herein is from about 0.05% to about 0.5% based on the total weight of the formulation. In one embodiment, the amount of formic acid in the formulations provided herein is from about 0.05% to about 0.1% based on the total weight of the formulation. In one embodiment, the amount of formic acid in the formulations provided herein is about 0.05%, 0.07%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5% based on the total weight of the formulation. In one embodiment, the formulations provided herein do not contain any formic acid. In one embodiment, the amount of formic acid in the formulations provided herein is from about 0.05% to 0.09% based on the total weight of the formulation.

Another aspect is a formulation comprising formic acid in an amount of no more than about 1mg in a 20-cc vial. Another aspect is a formulation comprising formic acid in an amount of up to about 0.2, 05, 0.7, 0.9mg, or 1mg in a 20-cc vial. Another aspect is a formulation comprising formic acid in an amount of about 0.3-0.9mg or 0.4 to 0.8mg in a 20-cc vial.

In another aspect, provided herein are formulations comprising compound D in an amount of about 1mg and HPBCD in an amount of about 800mg in a 20-cc vial.

In another aspect, provided herein are formulations comprising compound D in an amount of about 1mg, HPBCD in an amount of about 800mg, and formic acid in an amount of about 0.9mg in a 20-cc vial.

Formulation Ic

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.01% to 0.08% and HPBCD in an amount of about 99.40% to 99.99% based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.01% to 0.08%, HPBCD in an amount of about 99.40% to 99.99%, and no more than about 0.5% formic acid, based on the total weight of the formulation.

In one embodiment, provided herein are formulations comprising compound D in an amount of about 0.03% to 0.06% and HPBCD in an amount of about 99.60% to 99.99% based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising about 0.01% to about 0.08% of compound D, about 99.40% to about 99.99% hydroxypropyl β -cyclodextrin, and about 0.1% to about 0.3% formic acid, based on the total weight of the formulation.

In one aspect, the formulations provided herein comprise compound D in an amount of about 0.02% to about 0.06%, based on the total weight of the formulation. In certain embodiments, the amount of compound D is about 0.03% to about 0.06% or about 0.04% to about 0.06%, based on the total weight of the formulation. In certain embodiments, the amount of compound D is about 0.03%, 0.04%, 0.05%, or 0.06%, based on the total weight of the formulation. In one embodiment, the amount of compound D in the formulation is about 0.05% based on the total weight of the formulation.

In another aspect, provided herein are formulations comprising compound D in an amount of about 0.75mg to about 1.5mg in a 20-cc vial. Yet another aspect is the inclusion of compound D in an amount of about 0.75mg to about 1.25mg in a 20-cc vial. In one aspect, compound D is present in an amount of about 0.75, 0.8, 0.9, 1.0, 1.05, or 1.2mg in a 20-cc vial. In one aspect, compound D is present in an amount of about 1mg in a 20-cc vial.

In one embodiment, the amount of HPBCD in the formulations provided herein is about 99.40% to about 99.99% based on the total weight of the formulation. In one embodiment, the amount of HPBCD in the formulations provided herein is about 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, or 99.99% based on the total weight of the formulation. Another aspect is a formulation comprising HPBCD in an amount of about 1800-1900mg in a 20-cc vial. Another aspect is a formulation comprising HPBCD in an amount of about 1850 to 1900mg in a 20-cc vial. Another aspect is a formulation comprising HPBCD in an amount of about 1875mg in a 20-cc vial.

In one embodiment, the formulation comprises no more than about 0.5% formic acid, based on the total weight of the formulation. In one embodiment, the formulation comprises formic acid in an amount up to about 0.05%, 0.07%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5% based on the total weight of the formulation. In one embodiment, the formulation comprises no more than about 0.05%, 0.07%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5% formic acid, based on the total weight of the formulation. In one embodiment, the amount of formic acid in the formulations provided herein is from about 0.05% to about 0.3% based on the total weight of the formulation. In one embodiment, the amount of formic acid in the formulations provided herein is from about 0.05% to about 0.25% based on the total weight of the formulation. In one embodiment, the amount of formic acid in the formulations provided herein is about 0.05%, 0.07%, 0.09%, 0.1%, 0.2%, or 0.3% based on the total weight of the formulation. In one embodiment, the formulations provided herein do not contain any formic acid. In one embodiment, the amount of formic acid in the formulations provided herein is from about 0.11% to 0.3% based on the total weight of the formulation.

Another aspect is a formulation comprising formic acid in an amount of no more than about 4mg in a 20-cc vial. Another aspect is a formulation comprising formic acid in an amount of up to about 1, 1.8, 2, 2.1, 2.5, 3, 3.5, 3.8, 3.9, 4, 4.5, 4.9mg, or 5mg in a 20-cc vial. Another aspect is a formulation comprising formic acid in an amount of about 1 to 1.8mg, 2.1 to 3.8mg, or 3.9 to 4.9mg in a 20-cc vial.

In another aspect, provided herein are formulations comprising compound D in an amount of about 1mg and HPBCD in an amount of about 1875mg in a 20-cc vial.

In another aspect, provided herein are formulations comprising compound D in an amount of about 1mg, HPBCD in an amount of about 1875mg, and formic acid in an amount of about 2.1 to 3.8mg in a 20-cc vial.

Co-solvent free formulations

In one embodiment, provided herein are formulations comprising compound D in an amount of about 0.15% to 0.5%, citrate buffer in an amount of about 15% to about 35%, and HPBCD in an amount of about 92% to about 98%, based on the total weight of the formulation. In one embodiment, the citrate buffer includes anhydrous citric acid and anhydrous sodium citrate.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.25% to 0.30%, citrate buffer in an amount of about 30% to 32%, and HPBCD in an amount of about 67% to 69%, based on the total weight of the formulation.

In one embodiment, provided herein is a formulation comprising compound D in an amount of about 0.30% to 0.33%, citrate buffer in an amount of about 17% to 18%, and HPBCD in an amount of about 80% to 85%, based on the total weight of the formulation.

Exemplary formulations

In one embodiment, provided herein is a formulation consisting essentially of: compound D in an amount of about 0.05% to 0.25% and HPBCD in an amount of about 99.75% to 99.95% based on the total weight of the formulation.

In one embodiment, provided herein is a formulation consisting essentially of: compound D in an amount of about 0.05% to 0.25% and HPBCD in an amount of about 99.75% to 99.99% based on the total weight of the formulation.

In one embodiment, provided herein is a formulation consisting essentially of: compound D in an amount of about 0.05% to 0.25% and sulfobutyl ether- β -cyclodextrin in an amount of about 99.75% to 99.95% based on the total weight of the formulation.

In one aspect, provided herein is a formulation in a 20-cc vial, the formulation comprising: compound D in an amount to provide 1mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 800mg HPBCD; and about 0.6mg formic acid, as described herein. In one embodiment, the formulation in a 20-cc vial is reconstituted with 4.5mL sterile water for injection.

In one aspect, provided herein is a formulation in a 20-cc vial consisting essentially of: compound D in an amount to provide 1mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 800mg HPBCD; and about 0.6mg formic acid, as described herein. In one embodiment, the formulation in a 20-cc vial is reconstituted with 4.5mL sterile water for injection.

In one aspect, provided herein is a formulation in a 20-cc vial, the formulation consisting of: compound D in an amount to provide 1mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 800mg HPBCD; and about 0.6mg formic acid, as described herein. In one embodiment, the formulation in a 20-cc vial is reconstituted with 4.5mL sterile water for injection.

In one aspect, provided herein is a formulation in a 20-cc vial, the formulation comprising: compound D in an amount to provide 1mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 800mg sulfobutyl ether-beta-cyclodextrin; and about 0.6mg formic acid, as described herein. In one embodiment, the formulation in a 20-cc vial is reconstituted with 4.5mL sterile water for injection.

In one aspect, provided herein is a formulation in a 20-cc vial consisting essentially of: compound D in an amount to provide 1mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 800mg sulfobutyl ether-beta-cyclodextrin; and about 0.6mg formic acid, as described herein. In one embodiment, the formulation in a 20-cc vial is reconstituted with 4.5mL sterile water for injection.

In one aspect, provided herein is a formulation in a 20-cc vial, the formulation consisting of: compound D in an amount to provide 1mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 800mg sulfobutyl ether-beta-cyclodextrin; and about 0.6mg formic acid, as described herein. In one embodiment, the formulation in a 20-cc vial is reconstituted with 4.5mL sterile water for injection.

In one aspect, provided herein is a formulation in a 20-cc vial, the formulation comprising: compound D in an amount to provide 1mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 1875mg HPBCD; and about 2.1-3.8mg formic acid, as described herein. In one embodiment, the formulation in a 20-cc vial is reconstituted with 12.5mlL saline for injection.

In one aspect, provided herein is a formulation in a 20-cc vial consisting essentially of: compound D in an amount to provide 1mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 1875mg HPBCD; and about 2.1 to 3.8mg of formic acid, as described herein. In one embodiment, the formulation in a 20-cc vial is reconstituted with 12.5ml of normal saline for injection.

In one aspect, provided herein is a formulation in a 20-cc vial, the formulation consisting of: compound D in an amount to provide 1mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 1875mg HPBCD; and about 2.1 to 3.8mg of formic acid, as described herein. In one embodiment, the formulation in a 20-cc vial is reconstituted with 12.5ml of normal saline for injection.

In one embodiment, provided herein is an aqueous formulation comprising compound D in an amount of about 0.05% to 0.25% based on the total weight of solids, and HPBCD in an amount of about 99.1% to 99.9% based on the total weight of solids, and a diluent.

In one embodiment, provided herein is an aqueous formulation comprising compound D in an amount of about 0.05% to 0.25% based on the total weight of solids, and HPBCD in an amount of about 99.75% to 99.95% based on the total weight of solids, and a diluent.

In one embodiment, provided herein is an aqueous formulation consisting essentially of: compound D in an amount of about 0.05% to 0.25% based on the total weight of solids, and HPBCD in an amount of about 99.75% -99.95% based on the total weight of solids, and a diluent.

In one aspect, provided herein is an aqueous formulation comprising: compound D in an amount to provide 1mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 800mg HPBCD; about 0.6mg formic acid and about 4.5mL diluent.

In one aspect, provided herein is an aqueous formulation consisting of: compound D in an amount to provide 1mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; 800mg HPBCD; about 0.6mg formic acid and about 4.5mL diluent.

In one embodiment, provided herein is an aqueous formulation comprising compound D in an amount of about 0.01% to 0.08% based on the total weight of solids, and HPBCD in an amount of about 99.50% to 99.99% based on the total weight of solids, and a diluent.

In one embodiment, provided herein is an aqueous formulation consisting essentially of: compound D in an amount of about 0.01% to 0.08% based on the total weight of solids, and HPBCD in an amount of about 99.50% to 99.99% based on the total weight of solids, and a diluent.

In certain embodiments, the formulations provided herein are lyophilized, and the lyophilized formulations upon reconstitution have a pH of about 2.5 to 4. In certain embodiments, the lyophilized formulation after reconstitution has a pH of about 2.5 to 3.5. In certain embodiments, the lyophilized formulation after reconstitution has a pH of about 3.0 to 3.6. In one embodiment, the lyophilized formulation after reconstitution has a pH of about 2.5, 3, 3.2, 3.4, 3.6, 3.8, or 4. In one embodiment, the lyophilized formulation after reconstitution has a pH of about 2.5, 2.8, 3, 3.2, 3.4, 3.6, 3.8, or 4.

In certain embodiments, the lyophilized formulation after reconstitution has an osmolality of about 260-290 mOsm/kg. In certain embodiments, the lyophilized formulation after reconstitution has an osmolality of about 280 mOsm/kg. In certain embodiments, the lyophilized formulation after reconstitution has an osmolality of about 260 to 370 mOsm/kg. In certain embodiments, the lyophilized formulation after reconstitution has an osmolality of about 360 mOsm/kg. In certain embodiments, the lyophilized formulation after reconstitution has an osmolality of about 350 to 450 mOsm/kg. In certain embodiments, the lyophilized formulation after reconstitution has an osmolality of about 416 mOsm.

In certain embodiments, the lyophilized formulation is reconstituted with semi-physiological saline (sterile 0.45% sodium chloride solution for injection) and has an osmolality of about 280 to 320mOsm/kg upon reconstitution. In certain embodiments, the lyophilized formulation is reconstituted with semi-physiological saline (sterile 0.45% sodium chloride solution for injection) and, upon reconstitution, has a pH of 3.0 to 3.2 and an osmolality of about 280 to 320 mOsm/kg. In certain embodiments, the lyophilized formulation is reconstituted with 4.5mL of semi-physiological saline (0.45% sterile sodium chloride solution for injection) and has a pH of 3.0 to 3.2 and an osmolality of about 280 to 320mOsm/kg after reconstitution. In one embodiment, the required dose of the reconstituted solution is diluted to a volume of 50mL with physiological saline in an infusion bag (0.9% sodium chloride sterile solution for injection) for 30 minutes of intravenous administration.

In certain embodiments, the lyophilized formulation is reconstituted with physiological saline and has an osmolality of about 440mOsm/kg upon reconstitution. In one embodiment, the desired dose of the reconstituted solution is diluted with physiological saline to a volume of 50mL to obtain an administration solution having an osmolality of about 310 to 380 mOsm/kg. In one embodiment, the desired dose of reconstituted solution is diluted with physiological saline to a volume of 50mL to obtain an administration solution having an osmolality of about 310 to 355 mOsm/kg. In one embodiment, the desired dose of the reconstituted solution is diluted with physiological saline to a volume of 50mL to obtain an administration solution having an osmolality of about 317 to 371 mOsm/kg. In one embodiment, the desired dose of the reconstituted solution is diluted with physiological saline to a volume of 50mL to obtain an administration solution having an osmolality of about 317 mOsm/kg. In one embodiment, the desired dose of the reconstituted solution is diluted with physiological saline to a volume of 50mL to obtain an administration solution having an osmolality of about 371 mOsm/kg. In one embodiment, the dosing solution has an osmolality of no more than 352 mOsm/kg. In one embodiment, a dosing solution with a 4.8mg dose of compound D has an osmolality of 352 mOsm/kg.

In one aspect, provided herein is a formulation in a 20-cc vial, the formulation comprising: compound D in an amount to provide 1mg 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide; and bulking agents, as described herein. In one embodiment, the formulation further comprises no more than about 5mg of formic acid as a residual solvent. In one embodiment, the formulation further comprises no more than about 4mg of formic acid as a residual solvent. In one embodiment, the formulation further comprises no more than about 3mg of formic acid as a residual solvent. In one embodiment, the formulation further comprises no more than about 2mg of formic acid as a residual solvent. In one embodiment, the formulation further comprises no more than about 1.5mg of formic acid as residual solvent. In one embodiment, the formulation further comprises no more than about 1mg of formic acid as a residual solvent. In one embodiment, the formulation further comprises no more than about 0.8mg of formic acid as residual solvent. In one embodiment, the formulation comprises from about 0.4mg to about 1.5mg, from about 0.5mg to about 1mg, or from about 0.5mg to about 0.9mg of formic acid as residual solvent. In one embodiment, the formulation comprises about 0.4mg, about 0.6mg, about 0.8mg, about 1mg, or about 1.5mg of formic acid as residual solvent. In one embodiment, the formulation comprises formic acid as residual solvent in an amount from about 1.0mg/mg to about 1.8mg/mg, from about 2.1mg/mg to about 3.8mg/mg, or from about 3.9mg/mg to about 4.9mg/mg of compound D.

The formulations of compound D provided herein can be administered to a patient in need thereof using standard therapeutic methods for delivering compound D, including but not limited to the methods described herein. In one embodiment, the formulations provided herein are reconstituted in a pharmaceutically acceptable solvent to produce a pharmaceutically acceptable solution, wherein the solution is administered (such as by intravenous injection) to a patient.

In one aspect, the formulations provided herein are lyophilized, and the lyophilized formulations are suitable for reconstitution with a suitable diluent to an appropriate concentration prior to administration. In one embodiment, the lyophilized formulation is stable at room temperature. In one embodiment, the lyophilized formulation is stable at room temperature for up to about 24 months. In one embodiment, the lyophilized formulation is stable at room temperature for up to about 24 months, up to about 18 months, up to about 12 months, up to about 6 months, up to about 3 months, or up to about 1 month. In one embodiment, the lyophilized formulation is stable for up to about 12 months, up to about 6 months, or up to about 3 months when stored under accelerated conditions of 40 ℃/75% RH.

Any pharmaceutically acceptable diluent can be used to reconstitute the lyophilized formulations provided herein for parenteral administration to a patient. Such diluents include, but are not limited to, sterile water for injection (SWFI), 5% dextrose in water (D5W), or a cosolvent system. Any amount of diluent may be used to reconstitute the lyophilized formulation such that a suitable injectable solution is prepared. Thus, the amount of diluent must be sufficient to dissolve the lyophilized formulation. In one embodiment, the lyophilized formulation is reconstituted with 1 to 5mL or 1 to 4mL of diluent to yield a final concentration of compound D of about 0.05 to 0.3mg/mL or about 0.15 to 0.25 mg/mL. In certain embodiments, the final concentration of compound D in the reconstituted solution is about 0.25 mg/mL. In certain embodiments, the final concentration of compound D in the reconstituted solution is about 0.20 mg/mL. In certain embodiments, the volume of reconstituted diluent is varied between 3mL and 5mL to produce a final concentration of 0.15 to 0.3 mg/mL. In certain embodiments, multiple vials may be used for reconstitution depending on the desired dose.

Reconstituted solutions of the lyophilized formulation may be stored and used for up to about 24 hours, about 12 hours, or about 8 hours. In one embodiment, after reconstitution, the aqueous reconstitution solution is stable at room temperature for about 1 to 24, 2 to 20, 2 to 15, 2 to 10 hours. In one embodiment, the aqueous reconstitution solution is stable at room temperature for up to about 20, 15, 12, 10, 8, 6, 4, or 2 hours after reconstitution. In some embodiments, the solution is used within 8 hours after preparation. In some embodiments, the solution is used within 5 hours after preparation. In some embodiments, the solution is used within 1 hour after preparation.

Method for producing a formulation

The formulations provided herein can be prepared by any method known in the art and as described herein, but all methods include the steps of: the active ingredient is combined with a pharmaceutically acceptable excipient, which constitutes one or more necessary ingredients (such as bulking agents and/or buffers).

In one aspect, the formulations provided herein are prepared by: compound D, bulking agent and citrate buffer are dissolved in water and dimethyl sulfoxide (DMSO) to obtain a solution and the solution is optionally lyophilized.

In one embodiment, the method for preparing a formulation comprises: dissolving HPBCD in citrate buffer to obtain a buffer solution, dissolving compound D in DMSO to obtain a premix, adding the premix to the buffer solution to obtain a solution; and optionally lyophilizing the solution to produce a lyophilized formulation.

In one embodiment, the method comprises dissolving Kleptose HPB in 20mM pH 4 to 4.5 citrate buffer to obtain a buffer solution, dissolving compound D in DMSO to obtain an active premix, adding the premix to the buffer solution to obtain a mixture, adding water to the mixture to obtain a bulk solution, filtering the bulk solution through one or more 0.45 μm and 0.22 μm filters to obtain a filtered solution, filling the filtered solution into vials, and lyophilizing the solution. In one embodiment, the solution is filtered through one 0.45 μm and two 0.22 μm filters. In one embodiment, the method comprises dissolving Kleptose HPB in 20mM pH 4.3 citrate buffer to obtain a buffer solution, dissolving compound D in DMSO to obtain an active premix, adding the premix to the buffer solution to obtain a mixture, adding water to the mixture to obtain a bulk solution, filtering the bulk solution through one 0.45 μm filter and two 0.22 μm filters to obtain a filtered solution, filling the filtered solution into 20-cc glass vials, and optionally lyophilizing the solution. In one embodiment, the vial is sealed under nitrogen after lyophilization.

In one aspect, the formulations provided herein are prepared by: dissolving compound D in formic acid to obtain a premix, dissolving HPBCD in water to obtain a solution, adding the premix to the solution to obtain a drug solution; and optionally lyophilizing the drug solution to produce a lyophilized formulation.

In one aspect, the formulations provided herein are prepared by: dissolving compound D in formic acid to obtain an active premix, dissolving Kleptose HPB in water to obtain Kleptose solution, adding the premix to the Kleptose solution to obtain a mixture, adding water to the mixture to obtain a bulk solution, filtering the bulk solution through one or more 0.45 μm and 0.22 μm filters to obtain a filtered solution, filling the filtered solution into vials, and lyophilizing the solution. In one embodiment, the solution is filtered through one 0.45 μm and two 0.22 μm filters. In one embodiment, the method comprises dissolving compound D in formic acid to obtain an active premix, dissolving Kleptose HPB in water to obtain Kleptose solution, adding the premix to the Kleptose solution to obtain a mixture, adding water to the mixture to obtain a bulk solution, filtering the bulk solution through one 0.45 μm and two 0.22 μm filters to obtain a filtered solution, filling the filtered solution into 20-cc glass vials, and lyophilizing the solution. In one embodiment, the vial is sealed under nitrogen after lyophilization.

In one aspect, the lyophilization process contains three stages: freezing, primary drying and secondary drying. The liquid formulation is converted into a lyophilized powder form by completely solidifying through a freezing stage, sublimating ice and solvent by primary drying, and desorbing residual moisture and solvent by secondary drying. The shelf temperature and chamber pressure in the primary and secondary drying are controlled to achieve the desired quality of the finished drug product. In one aspect of the method, the appearance and structure of the tortilla is characterized by visual inspection.

5.7. Reagent kit

In one aspect, provided herein is a kit for identifying a subject having cancer who is likely to respond to a therapeutic compound, the kit comprising means for determining the level of a genetic signature (e.g., LSC signature) in a sample that has been treated with a therapeutic compound, wherein the therapeutic compound (including compound D) is a compound described in section 5.5 above.

In one aspect, provided herein is a kit for treating cancer, the kit comprising means for determining the level of a genetic signature (e.g., LSC signature) in a sample that has been treated with a therapeutic compound, wherein the therapeutic compound (including compound D) is a compound described in section 5.5 above.

In yet another aspect, provided herein is a kit for monitoring the efficacy of a therapeutic compound in treating cancer in a subject, the kit comprising means for determining the level of a genetic signature (e.g., LSC signature) in a sample that has been treated with a therapeutic compound, wherein the therapeutic compound (including compound D) is a compound described in section 5.5 above.

In certain embodiments of the various kits provided herein, the therapeutic compound is compound D, or a stereoisomer or a mixture of stereoisomers thereof, a tautomer, a pharmaceutically acceptable salt, a solvate, an isotopologue, a prodrug, a hydrate, a co-crystal, a clathrate, or a polymorph thereof.

In certain embodiments, the cancer is a blood cancer. In one embodiment, the blood cancer is lymphoma. In another embodiment, the blood cancer is leukemia. In yet another embodiment, the blood cancer is MM. In a specific embodiment, the leukemia is ALL. In another specific embodiment, the leukemia is AML. In yet another specific embodiment, the leukemia is CLL. In yet another embodiment, the leukemia is CML. In some embodiments, the AML is relapsed. In certain embodiments, the AML is refractory. In other embodiments, the AML is resistant to conventional therapies.

In still other embodiments, the cancer is characterized by an increased level of a characteristic LSC. In still other embodiments, the LSC feature is a LSC feature described herein. In one embodiment, provided herein is a kit for treating cancer, characterized by increasing the level of a LSC signature described herein with a therapeutic compound. In one embodiment, provided herein is a kit for treating leukemia characterized by increasing the characteristic levels of LSCs described herein with a therapeutic compound. In another embodiment, provided herein is a kit for treating AML characterized by increasing the characteristic levels of LSCs described herein with a therapeutic compound.

In certain embodiments, the LSC signature comprises at least one gene selected from the group consisting of: AKR1C3, ARHGAP22, CD34, CDK6, CPXM1, DNMT3B, DPYSL3, EMP1, GPR56, KIAA0125, lamm 4B, MMRN1, NGFRAP1, NYNRIN, SMIM24, SOCS2, and ZBTB 46. In some embodiments, the LSC signature comprises two, three, four, five, six, seven, eight, nine, ten, twelve, fourteen, sixteen, or all genes selected from AKR1C3, ARHGAP22, CD34, CDK6, CPXM1, DNMT3B, DPYSL3, EMP1, GPR56, KIAA0125, lamm 4B, MMRN1, NGFRAP1, NYNRIN, SMIM24, SOCS2, and ZBTB 46. In a particular embodiment, the LSC feature is a LSC17 feature comprising AKR1C3, ARHGAP22, CD34, CDK6, CPXM1, DNMT3B, DPYSL3, EMP1, GPR56, KIAA0125, lamm 4B, MMRN1, NGFRAP1, NYNRIN, SMIM24, SOCS2, and ZBTB 46. In some embodiments, the LSC signature is a LSC4 or LSC4 signature provided herein, i.e., a genetic signature comprising 4 genes: TNFRSF4, SLC4A1, SLC7A7, and AIM 2. In other embodiments, the LSC signature is a LSC3 or LSC3 signature provided herein, i.e., a genetic signature comprising 3 genes: SLC4A1, SLC7A7 and AIM 2.

In certain embodiments, the level of the LSC characteristic in the sample is about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, or about 100-fold greater than the reference level of the LSC characteristic.

In certain embodiments of the various kits provided herein, the sample is obtained from a tumor biopsy, a lymph node biopsy, or a biopsy from bone marrow, spleen, liver, brain, or breast.

In certain embodiments, provided herein is a kit for detecting mRNA levels of one or more genes in a gene signature. In certain embodiments, the kit comprises one or more probes that specifically bind to mRNA of one or more genes in the gene signature. In certain embodiments, the kit further comprises a wash solution. In certain embodiments, the kit further comprises reagents for performing a hybridization assay, mRNA isolation or purification means, detection means, and positive and negative controls. In certain embodiments, the kit further comprises instructions for use of the kit. The kit may be customized for home use, clinical use, or research use.

In certain embodiments, provided herein is a kit for detecting the protein level of one or more genes in a gene signature. In certain embodiments, the kit comprises a strip coated with an antibody that recognizes a protein biomarker, a wash solution, reagents for performing the assay, protein isolation or purification means, detection means, and positive and negative controls. In certain embodiments, the kit further comprises instructions for use of the kit. The kit may be customized for home use, clinical use, or research use.

Such kits may use, for example, the form of test strips, membranes, chips, discs, test strips, filters, microspheres, slides, multiwell plates, or optical fibers. The solid support of the kit can be, for example, plastic, silicon, metal, resin, glass, membrane, particle, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, membrane, plate, or slide. The biological sample may be, for example, a cell culture, a cell line, a tissue, an organ, an organelle, a biological fluid, a blood sample, a urine sample, or a skin sample.

In another embodiment, the kit comprises a solid support; a nucleic acid attached to a support, wherein the nucleic acid is complementary to at least 20, 50, 100, 200, 350, or more bases of an mRNA; and means for detecting mRNA expression in a biological sample.

In a specific embodiment, the medicament or assay kit comprises a compound or a pharmaceutical composition thereof in a container and further comprises a component for isolating RNA in one or more containers. In another specific embodiment, the medicament or assay kit comprises the compound or pharmaceutical composition in a container and further comprises components for performing RT-PCR, qRT-PCR, deep sequencing or microarray in one or more containers.

In certain embodiments, the kits provided herein employ means for detecting biomarker expression by qRT-PCR, microarray, flow cytometry, or immunofluorescence. In other embodiments, the expression of the biomarker is measured by ELISA-based methods or other similar methods known in the art.

In another specific embodiment, the medicament or assay kit comprises the compound or pharmaceutical composition thereof in a container and further comprises a component for isolating the protein in one or more containers. In another specific embodiment, the medicament or assay kit comprises the compound or pharmaceutical composition in a container and further comprises components for performing flow cytometry or ELISA in one or more containers.

In another aspect, provided herein are kits for determining the level of a gene signature that provide the material necessary to measure the abundance of one or more gene products in a gene signature or subset of gene signatures provided herein (e.g., one, two, three, four, five or more genes). Such kits may contain materials and reagents required for measuring RNA or protein. In some embodiments, such kits comprise a microarray, wherein the microarray consists of: oligonucleotides and/or DNA and/or RNA fragments or any combination thereof that hybridize to one or more gene products in a gene signature or subset of gene signatures provided herein. In some embodiments, such kits may comprise primers for PCR of RNA products or cDNA copies of RNA products or both of a gene signature or subset of gene signatures. In some embodiments, such kits may comprise PCR primers and qPCR probes. In some embodiments, such kits may comprise a plurality of primers and a plurality of probes, some of which have different fluorophores, to allow simultaneous measurement of multiple gene products of a gene signature or subset of gene signatures provided herein. In some embodiments, such kits may further comprise materials and reagents for producing cDNA from RNA. In some embodiments, such kits may comprise antibodies specific for protein products of a gene signature or subset of gene signatures provided herein. Such kits may additionally comprise materials and reagents for isolating RNA and/or proteins from a biological sample. In addition, such kits may comprise materials and reagents for synthesizing cDNA from RNA isolated from a biological sample. In some embodiments, such kits may comprise a computer program product embedded on a computer readable medium for predicting whether a patient is clinically susceptible to a compound. In some embodiments, the kit may comprise a computer program product embedded on a computer readable medium, and instructions.

In some embodiments, such kits measure the expression of one or more nucleic acid products of a gene signature or subset of gene signatures provided herein. According to this embodiment, the kit may comprise materials and reagents necessary to measure the expression of a particular nucleic acid product of a gene signature or subset of gene signatures provided herein. For example, a microarray or RT-PCR kit can be produced for a particular condition and contain only those reagents and materials necessary to measure the levels of a particular RNA transcript of a gene signature or subset of gene signatures provided herein to predict whether a patient's hematological cancer is clinically susceptible to a compound. Alternatively, in some embodiments, the kit may comprise materials and reagents necessary to measure expression of a particular nucleic acid product of a gene in addition to the gene signature provided herein. For example, in certain embodiments, the kits comprise reagents and materials required to measure the expression levels of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more genes in the gene signatures provided herein in addition to the reagents and materials required to measure the expression levels of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, 30, 35, 40, 45, 50, or more genes in addition to the gene signatures provided herein. In other embodiments, the kits contain reagents and materials necessary to measure the expression levels of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more genes in the gene signatures provided herein, as well as 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, or more genes not in the gene signatures provided herein. In certain embodiments, the kit contains a means for measuring at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more genes in a gene signature provided herein and 1-10, 1-100 in a gene signature not provided herein, 1-150, 1-200, 1-300, 1-400, 1-500, 1-1000, 25-100, 25-200, 25-300, 25-400, 25-500, 25-1000, 100-150, 100-200, 100-300, 100-400, 100-500, 100-1000 or 500-1000 genes.

For nucleic acid microarray kits, the kit typically comprises probes attached to the surface of a solid support. In one such embodiment, the probe may be an oligonucleotide or a longer probe, including probes ranging from 150 nucleotides to 800 nucleotides in length. The probe may be labeled with a detectable label. In a specific embodiment, the probe is specific for one or more gene products of a biomarker provided herein. The microarray kit may contain instructions for performing the assay and methods for interpreting and analyzing the data resulting from performing the assay. In a specific embodiment, the kit comprises instructions for predicting whether a hematological cancer in a patient is clinically susceptible to a compound. The kit may also contain hybridization reagents and/or reagents necessary to detect the signal generated when the probe hybridizes to the target nucleic acid sequence. Typically, the materials and reagents used in the microarray kit are in one or more containers. Each component of the kit is typically in its own suitable container.

In certain embodiments, the nucleic acid microarray kit comprises materials and reagents required to measure the expression levels of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 30, 35, 40, 45, 50, or more genes or combinations thereof in the gene signatures provided herein in addition to the materials and reagents required to measure the expression levels of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more genes or combinations thereof in addition to those in the gene signatures provided herein. In other embodiments, the nucleic acid microarray kit contains materials and reagents necessary to measure the expression level of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more genes, or any combination thereof, of the gene signatures provided herein, as well as 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, or more genes other than those of the gene signatures provided herein. In another embodiment, a nucleic acid microarray kit comprises measuring at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more genes, or any combination thereof, of the gene signatures provided herein and 1-10, 1-100 of the gene signatures not provided herein, 1-150, 1-200, 1-300, 1-400, 1-500, 1-1000, 25-100, 25-200, 25-300, 25-400, 25-500, 25-1000, 100-150, 100-200, 100-300, 100-400, 100-500, 100-1000 or 500-1000 genes.

For quantitative PCR, the kit typically contains preselected primers specific for a particular nucleic acid sequence. The quantitative PCR kit may also contain enzymes suitable for amplifying nucleic acids (e.g., polymerases such as Taq polymerase), deoxynucleotides, and buffers required for the amplification reaction. The quantitative PCR kit may further comprise a probe specific for a nucleic acid sequence associated with or indicative of a disorder. The probes may or may not be labeled with fluorophores. The probe may or may not be labeled with a quencher molecule. In some embodiments, the quantitative PCR kit further comprises components suitable for reverse transcription of RNA, including enzymes (e.g., reverse transcriptase, such as AMV, MMLV, etc.), and primers for reverse transcription, as well as deoxynucleotides and buffers required for the reverse transcription reaction. Each component of the quantitative PCR kit is typically in its own suitable container. Thus, these kits typically comprise different containers for each individual reagent, enzyme, primer and probe. In addition, the quantitative PCR kit may contain instructions for performing the reaction as well as methods for interpreting and analyzing the data generated by performing the reaction. In a specific embodiment, the kit contains instructions for predicting whether a hematological cancer in a patient is clinically susceptible to a compound.

For antibody-based kits, the kit can comprise, for example: (1) a first antibody that binds to a peptide, polypeptide, or protein of interest (which may or may not be attached to a solid support); and optionally, (2) a second, different antibody that binds to the first antibody or the peptide, polypeptide, or protein and is conjugated to a detectable label (e.g., a fluorescent label, a radioisotope, or an enzyme). In a particular embodiment, the peptide, polypeptide or protein of interest is associated with or indicative of a disorder (e.g., a disease). Antibody-based kits may also comprise beads for immunoprecipitation. Each component of the antibody-based kit is typically in its own suitable container. Thus, these kits typically comprise different containers for each antibody and reagent. In addition, antibody-based kits may contain instructions for performing the assay as well as methods for interpreting and analyzing the data resulting from performing the assay. In a specific embodiment, the kit contains instructions for predicting whether a hematological cancer in a patient is clinically susceptible to a compound.

In one embodiment, a kit provided herein comprises a compound provided herein, or a pharmaceutically acceptable salt, solvate, stereoisomer, isotopologue, prodrug, hydrate, co-crystal, clathrate, or polymorph thereof. The kit may further comprise additional active agents, including but not limited to those disclosed herein.

The kits provided herein can further comprise a device for administering the active ingredient. Examples of such devices include, but are not limited to, syringes, drip bags, patches, and inhalers.

The kit may further comprise cells or blood for transplantation and a pharmaceutically acceptable carrier useful for administering one or more active ingredients. For example, if the active ingredient is provided in a solid form that must be reconstituted for parenteral administration, the kit may comprise a sealed container of a suitable vehicle in which the active ingredient may be dissolved to form a sterile microparticle-free solution suitable for parenteral administration. Examples of pharmaceutically acceptable vehicles include, but are not limited to, water for injection USP; aqueous vehicles (such as, but not limited to, sodium chloride injection, ringer's injection, dextrose and sodium chloride injection, and lactated ringer's injection); water-miscible vehicles (such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol); and a non-aqueous vehicle (such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate).

In certain embodiments of the methods and kits provided herein, the solid support is used to purify a protein, label a sample, or perform a solid phase assay. Examples of solid phases suitable for performing the methods disclosed herein include beads, particles, colloids, single surfaces, tubes, multiwell plates, microtiter plates, slides, membranes, gels, and electrodes. When the solid phase is a particulate material (e.g., beads), in one embodiment it is distributed in the wells of a multiwell plate to allow parallel processing of the solid phase support.

It is noted that any combination of the embodiments listed above (e.g., with respect to one or more reagents, such as, but not limited to, nucleic acid primers, solid supports, etc.) is also contemplated with respect to any of the various methods and/or kits provided herein.

Certain embodiments of the present invention are illustrated by the following non-limiting examples.

6. Examples of the invention

Unless otherwise detailed, the following examples are carried out using standard techniques, which are well known and conventional to those skilled in the art. The described embodiments are intended to be illustrative only.

6.1. Leukemia Stem cell feature score

6.1.1.LSC17 score

Ng et al (Ng SW et al Nature.2016; 540(7633):433-37) previously reported a 17-gene score (LSC17 score) using a functional leukemia stem cell population, which showed that the LSC17 score is highly prognostic for a rapid determination of the risk and outcome of AML. More specifically, a high LSC17 score correlates with initial therapy resistance. Patients with a high LSC17 score have poor outcome with current treatments, including allogeneic stem cell transplantation. Therefore, LSC17 scores provide clinicians with a tool to identify AML patients who do not benefit from standard therapy according to Ng et al. As described in the examples below, the present disclosure is based, in part, on the surprising discovery of a particular correlation between the LSC17 score herein and responsiveness to compound D treatment.

The generation and description of LSC 17 scores is provided in more detail in the following paragraphs.

Based on the expression of CD34 and CD38, 83 cell samples obtained from 78 AML patients were sorted into fractions. Prkdc by xenograft to NOD ^scid .Il2rg ^null (NSG) mice to evaluate LSC activity in each fraction. Each of the 138 LSC + and 89 LSC-fractions, defined by function, was subjected to Gene Expression (GE) analysis. Obtaining a list of differentially expressed genes by comparing GE profiles of the LSC + and LSC-fractions; 104 genes showed 2-fold or more difference in expression level (P)<0.01). The LSC + reference profile is defined as the mean expression level of these 104 genes in the LSC + fraction.

To extract the dry core transcription component associated with clinical outcomes in a wide range of AML patient subtypes, a large dataset of 495 patients (Gene Expression integration (GEO) accession number GSE6891(Verhaak, r.g., haematologic 94, 131-.

Statistical regression algorithms were applied to link GE to patient survival in this training cohort based on the minimum absolute contraction and selection operator (LASSO) (Friedman, j., et al j.stat. softw.33,1-22 (2010); Simon, n., et al stat. softw.39,1-13(2011)) using a complete list of 89 LSC genes or a subset of 43 genes more highly expressed in the LSC + fraction.

Analysis of the latter subset yielded the best 17-gene signature (LSC17 score), which can be calculated for each patient as a weighted sum of 17 gene expressions, as shown in table 2 and the following algorithm:

LSC17 score for characteristics ═ (DNMT3B × 0.0874) + (ZBTB46 × 0.0347) + (NYNRIN × 0.00865) + (ARHGAP22 × -0.0138) + (lam 4B × 0.00582) + (MMRN1 × 0.0258) + (DPYSL3 × 0.0284) + (KIAA0125 × 0.0196) + (CDK6 × -0.0704) + (CPXM1 × -0.0258) + (SOCS2 × 0.0271) + (SMIM24 × -0.0226) + (EMP1 × 0.0146) + (NGFRAP1 × 0.0465) + (CD34 × 0.0338) + (AKR1C3 × -0.0402) + (GPR56 × 0.0501).

Since scores above and below the median in the training cohort were associated with unfavorable and favorable cytogenetic risks, respectively, median thresholds were used to discretize the scores into high and low groups.

Table 2: corresponding weights in LSC signature genes and LSC17 scores

LSC characteristic gene	Weight of
		DNMT3B	0.0874
ZBTB46	-0.0347
		NYNRIN	0.00865
ARHGAP22	-0.0138
		LAPTM4B	0.00582
MMRN1	0.0258
		DPYSL3	0.0284
KIAA0125	0.0196
		CDK6	-0.0704
CPXM1	-0.0258
		SOCS2	0.0271
SMIM24	-0.0226
		EMP1	0.0146
NGFRAP1	0.0465
		CD34	0.0338
AKR1C3	-0.0402
		GPR56	0.0501

To calculate the LSC17 score, leukemia cells may be collected from the patient's peripheral blood. The patient cells can be subjected to RNA-Seq to characterize gene expression profiles. In parallel to RNA-Seq, RNA extracted from patient samples can be evaluated in a NanoString assay to determine LSC17 scores. In certain embodiments, patient samples with LSC17 scores above the median threshold are assigned a high LSC17 score group, while patient samples with LSC17 scores below the median threshold are assigned a low LSC17 score group.

6.2. Efficacy of Compound D on Primary acute myeloid leukemia samples with different leukemia Stem cell characteristic scores

The following are assay examples that can be used to i) evaluate the efficacy and mechanism of action of compound D on preclinical models from one patient derived AML sample using in vitro and in vivo methods; ii) assessing the correlation of LSC17 score with compound D efficacy; and iii) assessing the differential effect on Leukemic Stem Cells (LSCs) and normal Hematopoietic Stem Cells (HSCs) by a secondary engraftment model.

6.2.1. Materials and methods

6.2.1.1. Test animals

The NOD/SCID mice used in this study were 10 week old females with an average body weight of 20 grams at the beginning of dosing.

6.2.1.2. Cell lines/cell cultures

All samples were tested for engraftment in NOD/SCID mice prior to use in efficacy studies.

Materials for in vitro determination of AML cells include X-VIVO 10 medium supplemented with 15% BIT and growth factors, including 100ng/mL stem cell factor, 20ng/mL Interleukin (IL) -6, 20ng/mL granulocyte colony stimulating factor (G-CSF), 20ng/mL IL-3,100ng/mL fms-like tyrosine kinase (Flt 3) ligand (each provided by Amgen in USA), 20ng/mL granulocyte-monocyte colony stimulating factor (GM-CSF; R & D Systems, USA), and 50ng/mL thrombopoietin (Kirin Brewer, Japan).

For AML Colony Forming Unit (CFU) assays, a 0.9% methylcellulose semisolid culture containing 15% Fetal Calf Serum (FCS), 15% pre-tested human plasma, 50. mu.M β -mercaptoethanol, and cytokines, stem cell factor at a concentration of 100ng/mL, 100ng/mL Flt-3 ligand, 20ng/mL IL-6, 20ng/mL GM-CSF, 20ng/mL IL-3, and 3U/mL erythropoietin (Amgen, USA) was used.

6.2.1.3. Assay materials and reagents

Apoptosis was assessed using the annexin V-PE apoptosis detection kit (BD Pharmingen, BD Bioscience, usa).

The following mouse anti-human antibodies were used to evaluate the efficacy of compound D in AML xenograft models (both from BD Biosciences, usa, unless otherwise noted): mouse anti-human CD45-APC, CD33-PC5.5(Beckman Coulter, USA), CD19-V450, CD14-PE, CD15-FITC, CD34 APC-Cy7 and CD38 PE Cy 7. Propidium iodide (BD, USA) was used to identify dead cells in the assay.

6.2.2. Design of experimental study

In this study, Leukemia cells were collected from peripheral blood of patients at Margaret Leukemia Bank and subjected to Ficoll gradient centrifugation to obtain monocytes for live cryopreservation. All samples were tested for engraftment in NOD/SCID mice prior to use in the study. Acute myeloid leukemia cells were used in short term in vitro suspension cultures (4 and 24 hours) to evaluate the effect of compound D on gstt 1 degradation and apoptosis, AML-CFU assay to evaluate the effect of compound D on colony forming progenitor cells, and xenografts were transplanted into NOD/SCID mice to evaluate the in vivo effect of compound D on AML. After completion of dosing, all animals were euthanized as planned the next day after the last compound D dose, and bone marrow was collected from the injected right femur and the non-injected left femur for flow cytometry analysis using human-specific antibodies to assess implantation. Secondary transplantation was also performed using Limiting Dilution Assay (LDA) to investigate whether compound D targets leukemic stem cells with self-renewal capacity.

6.2.3. Experimental procedures

6.2.3.1. In vitro degradation of Compound D G1 to S phase transition protein 1(GSPT1) and apoptosis assay

Preparation of test article stock solutions and dilutions: compound D was prepared in anhydrous DMSO to make 1M stock solutions, which were then further diluted to final concentrations of 3, 30, and 100 nM.

And (3) cell culture: materials for in vitro determination of AML cells include X-VIVO 10 medium supplemented with 15% BIT and growth factors including 100ng/mL stem cell factor, 20ng/mL IL-6, 20ng/mL G-CSF, 20ng/mL IL-3,100ng/mL Flt 3 ligand (each supplied by Amgen in the U.S.A.), 20ng/mL GM-CSF (R & D Systems, USA), and 50ng/mL thrombopoietin (Kirin Brewery, Japan).

Measurement procedure: after 4 and 24 hours in vitro culture with DMSO or compound D, cells were harvested for GSPT1 expression and apoptosis. Levels of GSPT1 were analyzed by flow cytometry using Median Fluorescence Intensity (MFI) and normalized against DMSO control. Apoptosis was also analyzed by flow cytometry and measured as the percentage of cells positive for cleaved caspase 3/7.

6.2.3.2. Acute myeloid leukemia-colony Forming Unit assay

Preparation of test article stock solutions and dilutions: compound D was prepared as follows.

Cell culture: acute myeloid leukemia cells were plated in 0.9% methylcellulose containing 15% FCS, 15% pre-tested human plasma, 50. mu.M β -mercaptoethanol, and cytokines, stem cell factor at a concentration of 100ng/mL, Flt-3 ligand at 100ng/mL, IL-6 at 20ng/mL, GM-CSF at 20ng/mL, IL-3 at 20ng/mL, and erythropoietin at 3U/mL (Amgen, USA). During suspension and CFU assays, acute myeloid leukemia cells were cultured with DMSO or compound D (prepared as described below).

Measurement procedure: after incubation of AML cultures with DMSO or compound D for 12 to 14 days at 37 ℃, plates were assigned a score for the presence of AML-CFU (CFU defined as >50 cells).

6.2.3.3. Xenograft assay

Preparation of test articles for drug delivery: for compound D administration in vivo, compound D was formulated according to the following protocol immediately prior to each administration.

Preparation: 5% NMP/45% polyethylene glycol (PEG) 400/50% saline; NMP-catalog No. 69118 (New accession No. M79603-1L), Fluka; PEG 400-Cat No. 81172-1L, Fluka; brine-0.9% sodium chloride.

Preparation: the desired amount of compound was weighed into a glass vial. Add NMP and vortex. Ensure that the entire compound is wet. PEG400 was added and vortexed until the solution was clear and particle free. Saline was added slowly and mixed thoroughly with a hand-held homogenizer with a disposable tip for about one minute. Immediately used, as the compound was unstable in the formulation over time.

Compound administration: compound D was administered by the intraperitoneal route. Vehicle and compound D were administered in a volume of 2.5mL/kg, twice daily (BID) (if tested once daily [ QD ] dosing, a dose volume of 5mL/kg was used). A twice daily regimen is recommended with 3 hours intervals between doses. Since the compounds are unstable over time in the formulation, they are formulated fresh for each application. Dose levels of 5mg/kg BID are not to be exceeded as this would result in a maximum concentration Cmax which may not be clinically relevant.

Femoral internal transplantation: one day prior to transplantation, NOD/SCID mice were preconditioned by sublethal irradiation (275cGy) and then injected with anti-CD 122 antibody (200 μ g/mouse) to deplete residual host Natural Killer (NK) cells. On the day of transplantation, live frozen AML bulk cells (see section 6.2.1.2) were thawed, counted, and transplanted intrafemora into pre-conditioned mice at a dose of 5 × 106 cells/mouse in a total volume of 30 μ Ι _ of phosphate buffered saline.

Treatment and assay procedures: on day 21 after AML transplantation, mice were randomized and given 2.5mg/kg of compound D or vehicle (5% N-methyl-2-pyrrolidone [ NMP ]/45% polyethylene glycol [ PEG ] 400/50% saline) intraperitoneally twice daily in a dose volume of 50 μ Ι _, for 4 weeks. All animals were euthanized at the planned termination time (1 day after the last treatment) and bone marrow was collected from the right femur (injected bone marrow) and left femur and left and right tibia (non-injected bone marrow). Cells isolated from injected or non-injected bone marrow were analyzed by flow cytometry to assess AML engraftment, and live frozen for future secondary engraftment analysis.

Cells harvested from injected and non-injected bone marrow were stained with mouse anti-human antibody as indicated in section 6.2.1.3. After staining, the washed cells were run on a LSRII flow cytometer (BD, usa) with 10,000 to 20,000 events collected per sample. The collected data were analyzed by FlowJo software (TreeStar, usa) to assess AML engraftment levels in different tissues, as determined by the percentage of human CD45+ CD33+ cells.

The normal cord blood experiment was performed similarly as described above, but using CD34+ cells isolated from normal cord blood. Two or three normal donors were pulled together to generate enough cells for each CB implant, termed CB1 and CB 2.

Secondary xenograft limiting dilution assay: to determine whether compound D targets leukemic stem cells with self-renewal capacity, which is thought to contribute to leukemia progression, therapy resistance and relapse, secondary transplants were performed using LDA. Limiting dilution assays were designed to define the unknown frequency of LSCs in total leukemia grafts in one mouse. LDA analysis in secondary transplantation will allow quantitative determination of whether compound D targets LSCs with self-renewal capacity in primary mice. For this, multiple cell doses were used for secondary transplantation to achieve positive responses (transplanted mice, high cell dose) and negative responses (non-transplanted mice, lowest cell dose). There were four different AML cell doses (million, 500,000, 50,000, and 2000 cells/mouse) per treatment group, with 5 mice per cell dose, and a total of 40 mice per AML graft sample. LDA was performed at lower cell doses for any sample considered aggressive. The frequency of LSCs was analyzed using the Walter and Eliza Hall Institute (WEHI) bioinformatics limiting dilution analysis (ELDA) software (bio if.

For secondary transplantation, NOD/SCID mice were sublethally irradiated (275cGy) and pre-treated with anti-CD 122 antibody (200. mu.g/mouse) to deplete residual host natural killer cells. On the day of transplantation, viable frozen cells harvested from vehicle or compound D treated primary mice were thawed, counted, subjected to mouse cell depletion (mouse cell depletion kit, Miltenyi Biotec, usa), and transplanted intrafemora into pretreated secondary mice at limited doses as described above. For LDA-free secondary transplantation, thawed cells did not deplete mouse cells prior to transplantation. Mice were euthanized 12 weeks after the secondary transplantation, and bone marrow was collected and analyzed.

6.2.3.4. Data analysis

Implantation of AML cells in injected femur and non-injected femur and tibia was analyzed by flow cytometry. Graph and statistical analysis were generated by GraphPad Prism software. Statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Tukey multiple post-comparison tests.

6.2.4. Effect of Compound D on in vitro GSPT1 degradation in acute myeloid leukemia cells

Ten primary AML patient samples were tested in vitro to investigate whether primary AML cells were sensitive to compound D and whether sensitivity to compound D varied in the AML samples. The level of GSPT1 in AML cells was first studied because compound D inhibits cell viability via degradation of GSPT 1. Compound D reduced the level of GSPT1 as early as 4 hours post-treatment, while GSPT1 levels remained low at 24 hours, compared to the control (fig. 1A-1B). The effect of compound D on the reduction of GSPT1 levels was concentration dependent. However, even at the highest compound D concentration (100nM), the level of degradation of GSPT1 varied from AML sample to AML sample. AML samples treated in vitro with compound D were grouped based on the LSC17 score described above. Surprisingly, the results show that the sample with a high LSC17 score has significantly higher degradation of GSPT1 compared to the sample with a low LSC17 score (fig. 1C).

Evaluation of inhibition of leukemia cell growth by apoptosis by compound D by flow cytometry. Acute myeloid leukemia cells were cultured with compound D for 24 hours. A representative flow cytometry analysis of apoptosis from 3 individual samples is shown in fig. 2A. No apoptosis was observed at 4 hours for all samples, whereas at 24 hours induction of apoptosis by compound D was observed in 3 out of 10 samples tested in a concentration-dependent manner (fig. 2B). Consistent with the GSPT1 degradation data, compound D induced apoptosis at a higher level in samples with a high LSC17 score compared to samples with a low LSC17 score (fig. 2B). Also consistent with apoptosis, cell counts showed that compound D decreased the number of cells in most samples with increasing concentration (fig. 2C). Cells from the sample with the higher LSC17 score had a greater reduction in cell number compared to the control compared to the sample with the lower LSC17 score (fig. 2C).

Colony formation assays were also performed to determine if primary leukemia cells were also sensitive to compound D. Of the 10 samples tested, 7 samples formed colonies, and the colony formation of all 7 samples decreased with increasing concentration of compound D (fig. 3). Of the 7 samples, 3 had a high LSC17 score and 3 other samples had a low LSC17 score. Compound D reduced colony formation more for the sample with a high LSC17 score than for the sample with a low LSC17 sample (fig. 3).

Taken together, these data indicate that compound D has an inhibitory effect on leukemic blasts and primary colony forming cells. Samples with high scores were more sensitive to compound D than samples with low LSC17 scores, as assessed by the degree of degradation of GSPT1, the level of apoptosis induction, the reduction in blast cell number, and the colony forming ability.

6.2.5. In vivo study to determine the Effect of dose assessment of Compound D on acute myeloid leukemia cells

Pilot studies were first performed to determine potential doses that could be used to target one AML graft in NOD/SCID mice. Two AML patient samples (AML 110500 and AML 90191) were tested intraperitoneally at 1.25 or 2.5mg/kg of compound D once daily (QD) or twice daily (BID), for a total of 4 different doses/schedule groups (figure 4). After pretreatment with sublethal radiation and anti-CD 122 antibody, no/SCID mice were intrafemora transplanted with AML cells previously harvested from the patient and live frozen. Starting on day 21, mice were administered compound D2 weeks after transplantation. As shown in figure 4 (top left panel), compound D reduced AML engraftment of AML110500 cells in patient samples in a dose-dependent manner relative to vehicle control. Acute myeloid lymphoma grafts in both Right Femur (RF) and uninjected Bone Marrow (BM) were significantly reduced at 2.5mg/kg QD and BID compound D, and the reduction of AML graft was greatest at 2.5mg/kg BID. CD34+ positive primitive leukemic cells were also most reduced by compound D in both RF and BM in mice receiving 2.5mg/kg BID compound D (fig. 4, left middle). The percentage of cells positive for the myeloid cell marker CD15 was also elevated in both RF and BM in mice dosed with 2.5mg/kg BID compound D (figure 4, bottom left). Notably, transplanted cells from patient sample AML 90191 were largely unaffected by compound D (fig. 4, right panel). Based on this pilot study, compound D was administered at 2.5mg/kg BID for the remainder of the in vivo experiments.

6.2.6. Efficacy of Compound D on acute myeloid leukemia grafts in xenografted mice

Six AML samples were selected for in vivo studies based on LSC17 scores, with 3 samples each with high and low scores. Clinical characteristics and other information of the samples used for the study are summarized in table 3. Two additional samples without LSC17 data were included in the assay subset.

Based on the pilot data (fig. 4), where one of the 2 samples responded to compound D administration at 2.5mg/kg BID for 2 weeks, the duration of administration was extended to 4 weeks to determine whether the mice could tolerate longer compound D administration. The clinical status of mice transplanted with 3 different AML samples was closely monitored during the treatment. Compound D treated mice were not sick and lost body weight after 1, 2 or 3 weeks of compound D treatment when compared to vehicle treated mice (table 4). Only one compound D-treated mouse was found paralyzed on the last day of treatment and death was found the next morning before scheduled sacrifice. This mouse was transplanted with AML cells from patient sample 120860, which responded poorly to compound D (fig. 6) and could die from a high leukemic burden and deep infiltration in case of antemortem paralysis.

Table 4: body weight of human acute myeloid leukemia xenografted mice after administration of Compound D

Avg is the average (mean).

In 6 AML samples tested for efficacy of compound D in vivo, cells from AML patient 90668 resulted in paralysis of the transplanted mice (5 million cells per mouse) before compound D treatment (day 21) began. Compound D treatment did not rescue these mice from paralysis or death. Thus, for AML patient 90668, the experiment was repeated in which fewer cells were transplanted. Even though there were 10-fold fewer AML cells transplanted (500,000 cells per mouse), the mice remained paralyzed around 4 weeks after transplantation. Administration of compound D from day 21 did not improve survival of mice from these transplanted AML patients 90668, probably because this sample was very aggressive in NOD/SCID mice at high engraftment levels and rapidly infiltrated into other organs as evidenced by rapid paralysis and splenomegaly of leukemic cell invasion.

For the other 5 AML samples tested, implantation of mice was sufficient to assess the efficacy of compound D. Compound D had a significant effect on 4 of 5 AML samples. Three of the 4 responder samples scored high in terms of LSC17 characteristics. After administration of compound D, acute myeloid leukemia cells were completely eradicated to undetectable levels, assessed by both the percentage of human CD45+ leukemia grafts in RF and BM and the absolute number of leukemia cells (fig. 5, left). Since compound D completely eradicated AML grafts in all 3 patient samples, the percentage of CD34+ blasts in compound D-treated mice was unreliable (non-specific and autofluorescent events). The absolute number of naive CD34+ cells was also at a very low and undetectable level in compound D treated mice (fig. 5, right).

LSC17 scores were low for AML 120860 and AML 100348 in the patient samples. Compound D did not reduce the number of AML cells from patient 120860 in the injected femur. The number of AML cells was significantly reduced in non-injected BM compared to vehicle control, but the effect was limited compared to the reduction of LSC17 high grafts (see figure 6, upper panel). The percentage and number of CD34+ blasts in the leukemic grafts for this sample were also not reduced by compound D. Sample AML 100348 had a significant response to compound D treatment, as determined by the reduction of human CD45+ leukemia grafts in both RF and BM (figure 6, lower panel), however, there were some levels of residual blasts and CD34+ primitive leukemia cells in compound D treated mice. These results indicate that samples with a low LSC17 score are likely to be less responsive to compound D than samples with a high LSC17 score.

6.2.7. Effect of Compound D on leukemia Stem cell Implantation in Secondary transplantation

To determine whether compound D targets AML leukemia stem cells with self-renewal, cells harvested from RF and BM of mice administered 2.5mg/kg compound D BID or vehicle were combined for transplantation into secondary mice. After mouse cell depletion, LDA was performed to determine the frequency of LSCs in samples with residual AML cells after compound D administration to transplanted mice. For

patient samples AML

90191 and 110500, LDA was performed on cells from mice administered 2.5mg/kg of Compound D BID. When mice were sacrificed at approximately 12 weeks post-transplantation, secondary mice transplanted with AML 90191 cells isolated from compound D primary treated mice had no AML engraftment, indicating the absence of residual LSCs in the samples isolated from the primary treated mice. However, AML 110500 cells isolated from the primary-dosed mice were successfully implanted into secondary mice (fig. 7A). Secondary mice transplanted with cells from compound D-treated mice had much lower leukemic grafts than secondary mice receiving vehicle-treated cells. By LDA analysis, a more than 13-fold reduction in LSC frequency was observed in compound D-treated one-time mice (fig. 7A). Cells from mice transplanted with patient sample AML 120860 (samples with low LSC17 scores and no response to compound D in primary mice) were also successfully re-populated into secondary mice in a Limiting Dilution Assay (LDA). All mice transplanted with cells from mice treated once with vehicle or compound D were implanted even at the lowest cell dose (20,000 AML cells per mouse) (fig. 7B). Data from non-injected BM was used to calculate leukemic stem cell frequency. There was no difference in LSC frequency (calculated using data from non-injected BM) in vehicle-treated and compound D-treated mice, indicating that compound D did not target LSC of patient sample AML 120860 as a non-responder (fig. 7B). Another LSC 17-low sample AML 100348 had a significant response to compound D in one treatment mice (figure 6). When cells from the patient sample AML 100348 harvested from the primary mice were injected into secondary mice (7500 to 200,000 per mouse), none of the transplanted mice were implanted, even the cells harvested from the vehicle mice (fig. 7C). Only when each mouse was injected with 1 million cells from vehicle-treated mice, the secondary mice were re-populated, indicating that the number of cells transplanted for LDA was too low. The remaining 3 AML samples with high LSC17 scores (

AML

0590, 110102 and 110770) were not subjected to limiting dilution assay, since leukemia cells could hardly be detected in mouse RF and BM due to the high efficacy of compound D on these patient samples. Thus, for each patient sample, the combined BM cells without mouse cell depletion of each treatment group were equally divided into 5 mice per treatment group. The percentage and total number of human CD45+ leukemia cells in one mouse and the number of AML cells transplanted per mouse under each condition are summarized in fig. 7D. Secondary mice transplanted with AML110770 cells from mice administered once with vehicle or compound D were not reintegrated. This is probably because 1) the secondary mice transplanted too few human leukemia cells (121 ten thousand cells for vehicle control and 40 ten thousand cells for compound D treatment per mouse), and 2) the transplanted host mouse cells competed with human leukemia cells because there was no mouse cell depletion of these patient samples.

In contrast, secondary mice transplanted with AML 0590 or AML 110102 vehicle control cells had more engraftment of AML cells compared to AML 110770 (fig. 7D-7E). However, cells harvested from compound D-treated primary mice did not re-lodge in secondary mice, indicating that residual leukemic cells in compound D-treated mice are not rich in sufficient LSCs with self-renewal capacity. These results indicate that compound D also targets LSC of both AML 0590 and AML 110102.

6.2.8. Effect of Compound D on Normal cord blood-derived human transplants

CB samples were used to study the toxicity of compound D on normal hematopoietic cells. Mice were transplanted with two different CB samples (CB1 and CB2) and dosed intraperitoneally with 2.5mg/kg of compound D or vehicle control BID. Flow cytometry analysis of representative mice from the vehicle-treated group and compound D-treated group are shown in fig. 8A and fig. 8B, respectively. A quantitative summary is shown in figure 9.

As shown in figure 9A, although compound D significantly reduced CB engraftment for both samples, CB cells were still engrafted in most compound D-treated mice. Thus, there was less inhibition of normal CB grafts than their effect on AML responders, especially than samples with high LSC17 scores that were completely eradicated after compound D administration.

Study of the cell population most affected by compound D in CB grafts. Compound D treatment resulted in a significant reduction in human grafts, however, 5% to 10% of grafts remained after treatment (fig. 9A). This is in contrast to the almost complete elimination of the graft in the sensitive AML samples (figure 5). The number of CD19+ lymphocytes (typically the major cell population developing in immunodeficient NOD/SCID mice) was significantly reduced by compound D (fig. 9B, top panel). In contrast, the proportion of CD33+ myeloid lineage cells increased after compound D administration. Whereas the total number of total CB grafts was greatly reduced (fig. 9B), the absolute number of CD33+ cells did not reflect an increase in frequency. Similar results were observed for CD15+ and CD14+ differentiated cells (fig. 9C). Compound D did not reduce glycophorin a (GlyA) + CD 45-erythroid cells in CB1 transplanted mice, but did result in a non-significant reduction of GlyA + CD 45-erythroid cells in CB2 transplanted mice (fig. 9D).

Primitive hematopoietic cells (CD34+) in CB grafts were also analyzed. Compound D did not reduce the percentage of CD34+ cells compared to the results of AML responders, while the absolute number of CD34+ cells was significantly reduced (fig. 10A), due to the significant reduction in total CB grafts treated with compound D. Similar to CD34+ cells, the percentage of CD34+ CD 38-primary cells (population enriched in normal hematopoietic stem cells) were not specifically targeted by compound D (fig. 10B). Of the CD34+ population, only CD34+ CD19+ naive lymphoid cells were significantly reduced by compound D (fig. 10C). CD34+ CD33+ primitive myeloid cells were not targeted by compound D (fig. 10D), indicating that compound D specifically targets CD34+ CD19+ primitive lymphoid cells and results in lymphopenia.

6.2.9. Conclusion

Compound D induces dose-dependent apoptosis in AML patient samples in vitro by degradation of GSPT 1. Compound D reduces colony-forming AML progenitor cells in vitro. Overall, NOD/SCID mice transplanted with different AML samples had good tolerance to compound D. During the 4-week treatment period, there were no clinical symptoms of the patient, including weight loss. Of the 7 AML samples that completed the complete treatment plan (including 2 AML samples for pilot, treatment time 2 weeks), 5 samples responded to compound D in the human AML mouse xenograft model. Both samples did not respond to compound D, indicating a different sensitivity to compound D between AML samples. These data show that there is a large direct relationship between LSC17 score and sensitivity to compound D. Acute myeloid leukemia was more sensitive to compound D treatment for the sample with a high LSC17 score than for the sample with a low LSC17 score. This is determined by a number of parameters including the level of degradation of GSPT1, inhibition of cell growth that induces apoptosis, reduction of colony forming progenitor cells, and in vivo eradication of AML transplants. Secondary transplantation showed that LSCs in responder leukemia grafts were also targeted. LSCs from sample AML 120860 were not targeted, with low LSC17 gene signature scores and were non-responders. Serial transplantation can be performed with more patient samples in order to confirm the effect of compound D on LSCs with self-renewal capacity. Compound D also reduced normal cord blood hematopoietic transplants in mice, but to a lesser extent compared to AML responders. CD19+ lymphoid progenitor cells and lymphocytes in CB grafts are primarily targeted. In contrast, other human cell types in CB grafts are much less sensitive to compound D.

The data generated by both in vitro and in vivo treatments clearly show that compound D inhibits primary AML cell growth by degradation of gstt 1. In AML samples, there were multiple sensitivities to compound D. The results show that the sample with a high LSC17 score is more sensitive to compound D toxicity than the sample with a low LSC score. As discussed, LSC17 scores were previously found to be highly prognostic and accurately predict initial therapy resistance of AML, i.e., patients with high LSC17 scores had poor outcome in current treatments including allogeneic stem cell transplantation (Ng SW et al Nature.2016; 540(7633): 433-37). Thus, the current finding that samples with high LSC17 scores are more sensitive to compound D indicates that compound D can target refractory AML that is resistant to current chemotherapy. Furthermore, the observation that compound D had less effect on normal hematopoietic transplants than AML responders supports the efficacy of compound D in AML patients with a high LSC17 score.

6.3. Discovery of the reactivity of acute myeloid leukemia to Compound D and potential predictive biomarkers for Compound D efficacy

The following are assay examples that can be used to i) determine the ratio of efficacy to resistance of AML to compound D by performing experiments on larger AML samples; ii) identification of potential biomarkers that can predict AML response/resistance to compound D by RNA Seq analysis of AML patient samples.

6.3.1. Materials and methods

Detailed information on test animals, cell lines/cell cultures, and assay materials and reagents is provided in section 6.2.1.

6.3.2. Experimental procedures

6.3.2.1.RNA-Seq

Ribonucleic acid (RNA) was extracted from primary leukemia cells, quantified and identified using a bioanalyzer, and then RNA-Seq was run. A total of 33 patients diagnosed with AML were used for RNA-Seq analysis, including 2 samples (110500 and 90191) which were tested for the effect of compound D in the study described in section 6.1.

6.3.2.2. Nano String for LSC17 scoring

Ribonucleic acids extracted for RNA-Seq were also sent to Nano String analysis to determine LSC17 scores. Each sample used for NanoString was analyzed with 150ng RNA in 5. mu.L using elemental chemistry assays. Twenty samples known to have LSC17 high and low scores were subjected to NanoString analysis and used as controls.

6.3.2.3. Preparation of stock solutions and dilutions of Compound D

The procedure for preparing a solution of compound D for animal administration is described in section 6.2.3.3.

Stock solutions and dilutions for in vitro experiments were prepared as follows: compound D was first dissolved in anhydrous Dimethylsulfoxide (DMSO) to reach a concentration of 1M, and then further serially diluted to different concentrations (10mM, 10 μ M, 1 μ M) in the completed medium for cell culture. The final concentrations of compound D for in vitro culture were 3nM, 30nM and 100 nM.

6.3.2.4. In vivo efficacy of Compound D on AML

Immunodeficient NOD/SCID mice received sublethal irradiation (225cGy) one day prior to AML transplantation and were treated with anti-CD 122 antibody to eradicate residual mouse NK cells. AML cells were plated once at 5x10 from each patient ⁶ Cell dose per mouse injected intrafemora into the right femur of the mouse, where 10 mice were transplanted per sample. Compound D and vehicle treatment was started on day 21 post-transplantation. Compound D was administered Intraperitoneally (IP) at 2.5mg/kg twice daily, 3 hours apart, for 4 weeks. Compound D was dissolved freshly into the solution before each treatment. Vehicle is the same solution without compound D compound and control treated mice are given the same volume (50 μ Ι _ per mouse) with the same treatment plan as compound D treatment. For each patient sampleEach treatment group had 5 mice.

After treatment was completed, cells were harvested from both injected Right Femur (RF) and non-injected bone marrow (BM, including left femur, 2 tibiae) and stained with human antibodies to assess the level of implantation of AML. Antibodies used for staining include: mouse anti-human CD45-APC, CD15-FITC, CD34-APC7, CD38-PC7(BD Biosciences, USA), CD14-PE, CD33-PC5(Beckman Coulter, USA), CD19-V450, CD19-AF700, CD11b-APC7, CD34-BV421(BD Biosciences, USA), and propidium iodide (PI; Invitrogen, USA).

6.3.2.5. In vitro assay of the Effect of Compound D on the expression of GSPT1, apoptosis and colony growth of Primary leukemia cells

Live frozen primary leukemia cells were thawed and plated in suspension cultures in Iscove Modified Dulbecco's Medium) + 15% BIT serum replacement (Stem Cell Technology, canada) supplemented with various human growth factors. Compound D was added to the culture at the indicated concentration.

For GSPT1 expression, intracellular flow cytometry (FACS) was performed by staining cells with GSPT1 conjugated with Alexa Fluor 647 in 24 hours of culture. For apoptosis, cells were harvested at 24 hours in culture and stained with annexin V-PE and 7-amino-actinomycin D (7AAD) (BD Biosciences, USA).

Colony assays were performed in semi-solid cultures supplemented with growth factors in the presence of compound D or DMSO as controls. Colonies were counted on day 14.

6.3.2.6. In vivo effects of Compound D on GSPT1 expression in xenograft AML models

After 4 weeks of transplantation with AML cells, mice were treated with 2.5mg/kg of compound D twice daily for a total of 3 doses. Four hours after the last treatment, cells were harvested from both RF-injected and non-injected BM of each mouse and stained, fixed and permeabilized with CD45-FITC (BD Biosciences, usa). Cells were then stained with GSPT1-Alexa Fluor 647 for intracellular FACS to detect expression of GSPT1 in implanted leukemia cells.

6.3.2.7. Data analysis

Implantation of AML cells in injected and non-injected femurs was analyzed by flow cytometry. Graph and statistical analysis were generated by GraphPad Prism software. Statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Tukey multiple post-comparison tests.

6.3.3. Heterogeneous reaction of acute myeloid leukemia samples to Compound D

A total of 31 clinically characterized patient samples (table 5) were tested in a xenograft assay to determine the efficacy of compound D on AML in mice. Leukemia engraftment was assessed by the percentage of CD45+ CD33+ population in RF-injected and non-injected BM. Some samples were only re-populated into mouse RF at low levels and leukemic cells were very low or undetectable in the BM (120347, 130311, 5786 and 141104). Patient 90156 did not re-enter mouse RF or BM at the time of analysis. Other AML samples were implanted with injected RF and no BM (figure 11). Most of the implanted samples (24 out of 28) had a significant and dramatic response to compound D, consistent with previous pilot studies on compound D. In those reaction samples, compound D reduced the AML burden in both RF injected and non-injected BM, while leukemic cells in BM had a more profound response to compound D (figure 11). Some samples responded less and a few were resistant to compound D, indicating that while compound D was potent on AML, the reactivity was different in the AML samples.

Table 5: molecular characterization of samples from 31 patients with acute myeloid leukemia

AML ═ acute myeloid leukemia; dx-diagnosis; flt3-ITD ═ fms-like tyrosine kinase 3-internal tandem repeats; flt3-TKD ═ fms associated tyrosine kinase 3-tyrosine kinase domain; ID is identification; LPD ═ lymphoproliferative disorder; MDS ═ myelodysplastic syndrome; MPN ═ myeloproliferative tumors; MRC ═ myelodysplastic-related changes; NHL ═ non-hodgkin lymphoma; NPM1 ═ nucleophosmin 1.

6.3.4. Compound D induces differentiation of primitive acute myeloid leukemia cells

In the case where compound D was observed to have an attractive effect on mouse AML, the question of whether compound D targets primitive leukemia cells and induces differentiation was next investigated, as AML cells are immature blasts with differentiation and block of maturation in patients. The sample of interest is one that still has significant residual leukemia cells in mice after compound D treatment. As shown in fig. 12A, 3 representative samples had significantly increased expression of the myeloid differentiation marker CD15 after compound D treatment. Compound D also induced the expression of the monocyte marker CD14 in the transplant of patient 120287. The majority of cells harvested from mice transplanted with patient sample 120093 were CD34+ primary cells lacking CD15 expression. Compound D treatment induced CD15 expression and reduced the CD34+ cell population in parallel, indicating that compound D targets and differentiates CD34+ naive cells in this sample. Compound D also reduced CD34+ cells and reduced CD14+ or CD11B + cells in the grafts of patient samples 100348 and 130826 (fig. 12B). Unlike both samples, compound D only eliminated CD14+ cells from 100474 and had more CD34+ primary cells remaining in the remaining graft. CD11b is also a myeloid differentiation marker and is increased on patient 150250 transplanted cells, in parallel with increased expression of another myeloid differentiation marker, CD 15. However, compound D decreased CD11B + leukemia cells with the decrease in CD34+ cells of patient 130826 (fig. 12B). The changes in CD15, CD14, and CD34 positive populations are summarized in fig. 12C-12E, and 3 different patterns are evident following compound D treatment: increase, decrease, or no change in population in AML grafts. Samples with decreased CD34+ blasts and increased CD15+ and/or CD14+ cells were samples that responded well to compound D (such as

patient samples

110555, 110500, and 120093). Samples that did not or poorly respond to compound D had unchanged or even increased CD34+ blasts, while some samples that responded to compound D in their grafts also had increased CD34+ cells. No increase in expression of myeloid differentiation markers CD15 and CD14 was observed in samples that were resistant or poorly responsive to compound D. Current studies show that while compound D dramatically eliminates total AML transplants and thus the absolute number of both primitive and differentiated leukemic cells is significantly reduced, compound D targets primitive leukemic cells and leads to myeloid differentiation in at least some samples. A second transplantation may be performed to determine if compound D targets LSCs that are self-renewing.

6.3.5. Identification of biomarkers of response to compounds for treating acute myeloid leukemia

6.3.5.1. Relationship between Compound D reactivity and score obtained from Nano String

Acute myeloid leukemia is a group of hematological malignancies with phenotypic and genetic heterogeneity and its responsiveness to clinically induced therapies varies from patient to patient. To determine which patients responded better to compound D and whether compound D had an effect on samples resistant to clinical therapy, patient cells were next subjected to RNA-Seq to characterize the gene expression profile of the samples used for the study. In parallel to the RNA-Seq, RNA extracted from patient samples was also sent to a NanoString assay to determine the LSC17 score described above. A total of 33 AML samples (including 31 samples used in the current SRA amendment and 2 samples from a prior pilot SRA study) were subjected to NanoString analysis. Twenty samples for which LSC17 scores were previously determined were run in parallel as controls. Of the 33 AML samples analyzed, only 6 had a low LSC17 score (see table 6), probably due to the samples used having the criteria required to achieve the study: high implantation capacity of xenografts and large number of biological library vials. Such samples are typically characterized as being from invasive disease with poor outcome and therefore have a high LSC17 score.

Table 6: acute myeloid leukemia sample-NanoString for LSC17 score

ID is identification; LSC ═ leukemic stem cells; RNA-ribonucleic acid.

^a Some samples (120347, 130311, 5786, and 141104) were only re-populated into mouse RF (right femur injected) at low levels, and BM (bone marrow not injected) had very low or undetectable leukemia cells. Patient 90156 did not re-enter mouse RF or BM at the time of analysis.

^b Samples studied in the pilot SRA study were as described in section 6.1.

Table 7 presents the reference values for the classification of high and low LSC17 scores.

Table 7: reference value for NanoString classification

high score hi; ID is identification; and lo is low score.

As shown in fig. 12, there was a heterogeneous reaction between samples, which could be grouped into 3 subgroups (fig. 13). Implantation of mice resulted in less than 10% of the samples in vehicle-treated RF and BM tissues being considered too low for efficacy analysis and excluded. Seventeen samples had dramatic responses to compound D in their RF, as most leukemic cells were eradicated, with 14 samples showing similar responses in their BM (> 80% reduction, group 1). The other 10 samples had a convenient response, but in their RF were lower than in group 1, and in the BM the response of 6 samples was similar (50% to 75% reduction, group 2). The remaining 9 samples had poor response and the reduction in RF was less than 25%. 4 of 9 samples did not respond at all to compound D in the injected RF, and 3 of 6 samples did not reduce AML in the BM (group 3). The efficacy of compound D was next analyzed based on the classification of high and low LSC17 scores to determine whether they correlated with the reactivity of AML samples to compound D. While one sample with a low LSC17 score did not respond to compound D in the injected right femur and had less than a 25% reduction in the non-injected bone marrow, the other 7 samples with a low LSC17 score responded very well to compound D (more than a 50% reduction, fig. 13). 8 of the 9 non-responders in RF had high LSC17 scores, and 5 of the 8 non-reacted samples also reacted poorly in BM. Interestingly, most samples with a high LSC17 score (which should have a poor prognosis, resistance to clinical chemotherapy) responded well to compound D with a more than 50% reduction in leukemia. A large number of samples had a very impressive response with leukemia reduction over 80% (13 in RF and 17 in BM, fig. 13), indicating that compound D was very potent on AML cells from most samples with high LSC17 scores.

6.3.5.2. Gene expression biomarkers predicting AML response to Compound D

The gene expression profiles of patient samples generated from RNA-Seq were next analyzed to find biomarkers that could predict AML response to compound D. Twenty-six samples met the analysis conditions. The LSC17 scores and the correlation with the mean LSC + and LSC-gene expression profiles did not significantly correlate with the percentage of AML reduction, most likely because most of the samples used in the current study had high LSC scores and a high percentage of AML reduction caused by compound D. Thus, increasing the number of samples with low LSC scores will greatly increase the chances of detecting a significant trend.

Next, by using the percentage of AML reduction as a response to guide the selection of signature genes, an optimistic sub-score was sought that could predict the response to compound D. 75% of the samples (n-20) were used for training and the remaining 25% of the samples (n-6) were used for testing. When LSC17 and 43 LSC + genes (see section 6.1) were selected for training and testing, no features were found to predict response with high accuracy.

However, a 4-gene score was identified among 89 LSC genes (see section 6.1), which can predict the percent reduction in AML with moderate accuracy (fig. 14, panel a, r ═ 0.77, p ═ 0.10). The predictions obtained by the median of the scores for all samples used in this study were discretized into "response" or "no response" (cutoff for no response was 25% reduction) and also show a correlation between score and% reduction (fig. 14, panel B, r ═ 0.87, p ═ 0.02). The four signature genes and their normalized weights are shown in table 8, and the algorithm is as follows:

4-gene score (LSC4 signature score) (TNFRSF4 × -1.13) + (SLC4a1 × 13.59) + (SLC7a7 × -3.57) + (AIM2 × -3.04).

Positive weights indicate that higher expression of the relevant gene will increase the percent reduction in the experiment, while negative weights indicate that higher expression of the relevant gene will increase the percent reduction. TNFRSF4 is highly expressed in LSC + samples and the protein is expressed significantly more in relapses than in diagnostic samples, usually with a higher frequency of LSCs observed at relapse. The remaining 3 signature genes were highly expressed in the LSC samples.

Table 8: corresponding weights in LSC signature genes and 4-Gene scores

LSC ═ leukemic stem cells.

Since 3 of the 4 genes were highly expressed in the LSC-sample, the LSC-data were subsequently analyzed for predictor scores. Training the characteristics of 46 LSC-genes yielded 3-gene scores (see below) that predicted a reduction in AML, and the results were very similar when compared to the 4-gene scores described above. Indeed, the 3 genes that make up the 3-gene score are the 3 LSC-genes in the 4-gene score: SLC4a1, SLC7a7 and AIM2 (figure 14, panel C), indicating that the LSC-sample may respond better to compound D. The 3-gene score was as follows:

3-gene score (LSC3 trait score) ═ SLC4a1 × 13.59) + (SLC7a7 × -3.57) + (AIM2 × -3.04).

In conclusion, the 4-gene score and the 3-gene score correlated well with the response to compound D.

6.3.5.3. Clinical characterization of samples associated with Compound D response

The clinical characteristics of the samples were studied to determine if any clinical profile was associated with compound D response. Cells collected from secondary and relapsed AML patients had a similar compound D response in both RF and BM as cells from new AML patients (figure 15A). Patients with poor prognosis responded better than samples with moderate prognosis, without significant difference, based on median percentage of AML graft reduction in mice (figure 15B). Abnormal karyotype patients with generally poor prognosis also had better responses than normal karyotype samples (fig. 15C). Cytogenetically normal AML samples with FLt3-ITD had a slightly lower response to compound D in RF injection, but similar responses in BM, compared to wild type FLt3 samples with generally better prognosis (fig. 15D). Despite the limited number of patient samples analyzed here, the level of response to compound D in relapsed or secondary AML samples with aberrant cytogenetics and poor prognosis was similar to newly diagnosed AML samples with intermediate prognosis.

6.3.6. targeting and degradation of GSPT1 by Compound D in vivo

The underlying mechanism of the effect of compound D on AML is that compound D degrades the translation terminator GSPT1 by recruiting GSPT1 to cereblon in the E3 ubiquitin ligase complex. Next, it was investigated whether compound D treatment in vivo reduced gstt 1 in mouse AML cells and whether GSPT1 degradation was responsible for AML graft reduction. Prior to use in vivo treatment, some samples were tested in vitro to determine whether compound D could reduce GSPT1 in AML cells. Similar to previously performed pilot experiments, exposure of AML cells to compound D reduced GSPT1 expression (fig. 16A). Increased apoptosis was observed within 24 hours (fig. 16B), and viable cells were reduced (fig. 16C). Colony formation assay showed that compound D inhibited colony formation of leukemia progenitor cells (fig. 16D), indicating that compound D degraded GSPT1 in leukemia cells and inhibited proliferation of both leukemia cells and leukemia progenitor cells by inducing apoptosis.

Next, it was investigated whether compound D also induced apoptosis in xenografts. Harvested cells were stained with Propidium Iodide (PI) 4 weeks after compound D treatment to detect apoptotic and dead leukemic cells. The number of PI + events in the bone marrow of mice was significantly increased, indicating that compound D administration resulted in the induction of apoptosis and cell death (fig. 17). To determine whether GSPT1 could also be degraded by in vivo compound D administration, leukemia cells were harvested for intracellular flow cytometry after 3-dose compound D treatment to assess the level of GSPT1 in both injected RF and non-injected BM. In the majority of the 17 test samples (fig. 18A), a reduction in the level of GSPT1 was found in either RF or BM or in both RF and BM. It appears that compound D degrades GSPT1 more deeply in RF injection than in non-injected BM. More samples had a smaller reduction in gstt 1 in the non-injected BM than in RF. Perhaps this reflects a difference in niche or blood supply between these sites; intrafemoral (IF) injection involves reaming the femoral cavity prior to cell injection. The level of reduction of GSPT1 by compound D varied from sample to sample and was independent of the reactivity of leukemic cells to compound D (fig. 18B). While some samples that reacted well to compound D had a significant reduction in GSPT1 in both RF and BM (e.g., Pt120287 and 110555), other compound D reactors did not show a dramatic reduction in GSPT1 (e.g., Pt130607 and 150250). In some samples, the reduction in GSPT1 was different between RF and BM, for example,

Pt

130826 and 150238 had opposite GSPT1 responses in RF and BM. The complex observations of the reduction in gstt 1 after short-term compound D treatment may be due to heterogeneous reactions between AML samples. Compound D treatment of varying duration should be performed to accurately capture the in vivo reduction in gstt 1 for each AML sample. However, the results show that for most samples, GSPT1 can be degraded by compound D in vitro and in mice. However, more samples need to be tested to reach the conclusion: whether GSPT1 degradation could be a biomarker predictive of AML response to compound D.

6.3.7. Conclusion

Studies with 31 AML samples showed that cereblon modulator compound D was very potent on AML in preclinical mouse models (including samples with poor prognosis and high LSC17 score). Sub-scores in LSC-associated genes were found to predict the response of AML to compound D.

Patients with a high LSC17 score are generally resistant to standard leukemia chemotherapy and have a poor prognosis. Observations in the current study indicate that if AML patients with more aggressive disease in the context of primary induction therapy can be quickly identified (such as by using the LSC17 scoring method), compound D may be suitable for clinical trials as a new therapeutic for such patients. Further analysis of LSC-associated genes from RNA-Seq yielded a 4-gene set score that could predict the responsiveness of leukemia to compound D. Three of the 4 genes are LSCs ^- Genes with a predicted function similar to the 4-gene score.

In addition to compound D induced apoptosis and cell death, FACS analysis of AML cell phenotype after compound D treatment in mice showed that compound D also altered cell surface markers in some of the response samples, including myeloid differentiation markers CD15, CD14, CD11b, indicating that at least a portion of the treated patient samples were responsive to compound D and had the ability to induce leukemia differentiation. In parallel, compound D treatment also reduced the proportion of CD34+ blasts in AML grafts. Secondary transplantation experiments with Limiting Dilution Assay (LDA) described in section 6.4 can reveal whether compound D also targets functional LSCs capable of replicating AML in serial transplantation.

In summary, compound D has shown strong inhibitory effect on AML in preclinical mouse models of human AML through studies of the effect of cereblon modulator compound D on AML. These observations provide significant implications for compound D in future clinical trials to treat patients with poorer prognosis and chemotherapy resistance, and the results suggest that compound D can reduce the likelihood of AML relapse.

6.4. Study of Effect of Compound D on acute myeloid leukemia Stem cells Using Secondary transplantation limiting dilution assay

6.4.1. Materials and methods

6.4.1.1. Test animals

The NOD/SCID mice used in this study were 10 week old female mice with an average body weight of 20 grams at the beginning of treatment.

6.4.1.2. Cell lines/cells

All patient samples used in these studies were collected with informed consent from Princess Margaret leukomia Bank and subjected to Ficoll gradient centrifugation to obtain monocytes for live cryopreservation. All samples were tested for engraftment in NOD/SCID mice prior to use in the study. The day after the last compound D treatment, mice treated with vehicle or compound D were sacrificed. Cells were harvested separately from right femur (RF; AML cell injection) and uninjected bone marrow (BM: left femur + left and right tibia) and aliquoted for FACS analysis to assess the efficacy of compound D on AML transplants in mice. The remaining cells from the same tissue (RF or BM) from each treatment group were pooled and live frozen for future secondary transplantation. For secondary transplantation, frozen cells were carefully thawed, filtered to remove dead cells, and then human leukemia cells were purified by the Mouse Cell Depletion process (Mouse Cell Depletion Kit, catalog No. 130-. Purified cells were counted, diluted for LDA, and injected intrafemorially into irradiated secondary female NOD/SCID mice.

6.4.1.3. Assay materials and reagents

Human AML cells engrafted in NOD/SCID xenografts were identified by cell surface marker expression of human CD45(dim level) and CD 33. The following combination of anti-human antibodies was used to detect human AML cells in secondary xenografts: CD 45-allophycocyanin (APC; Cat. No. 340943, BD, USA), CD 33-phycoerythrin-cyanine 5(PE-Cy 5; Cat. No. PN IM2647U, Beckman Coulter, USA), CD19-V450 (Cat. No. 560353, BD Biosciences, USA), CD14-PE (Cat. No. PN IM0650U, Beckman Coulter, USA), CD 15-fluorescein isothiocyanate (FITC; Cat. No. 347423, BD, USA), CD34-APC-Cy7 (Cat. No. 624072, BD Biosciences, USA), and CD38-PE-Cy7 (Cat. No. 335790, BD, USA).

6.4.2. Design of experimental study

Secondary transplants were performed in this study including LDA to investigate whether compound D targets LSCs with self-renewal capacity in treated primary mice.

6.4.3. Experimental procedures

6.4.3.1. Secondary xenograft limiting dilution assay

Limiting dilution determination: limiting dilution assays were used to limit the frequency of leukemic stem cells in total leukemic transplants in one mouse. Therefore, LDA analysis in secondary transplantation will allow quantitative determination of whether compound D targets leukemic stem cells with self-renewal capacity in primary mice. For this, multiple cell doses were used for secondary transplantation to achieve positive responses (transplanted mice, high cell dose) and negative responses (non-transplanted mice, lowest cell dose). Four different AML cell doses (1 million, 500,000, 50,000 and 2000 cells/mouse) were used per treatment group, with 5 mice per cell dose, for a total of 40 mice per leukemia graft sample. LDA was performed at lower cell doses for any sample considered aggressive. The frequency of LSCs was analyzed using the Walter and Eliza Hall Institute (WEHI) bioinformatics limiting dilution analysis (ELDA) software (bio if.

Femoral internal transplantation: one day prior to transplantation, NOD/SCID mice were sublethally irradiated (275cGy) and pre-treated with anti-CD 122 antibody (200. mu.g/mouse) to deplete residual host natural killer cells. On the day of transplantation, live frozen cells harvested from mice treated once with combined vehicle or compound D (2.5mg/kg, intraperitoneally, twice daily for 14 days, part 6.1) were thawed, counted, mouse cells depleted, and transplanted intrafemora into pre-treated secondary mice in a limited dose of total volume of 30 μ Ι. Low compound D treatment samples (< 25%) were implanted for human AML, cells from both RF and BM were pooled and 2 rounds of mouse depletion were performed to ensure high human AML cell purity. After mouse cell depletion, flow cytometry assays on a LSRII flow cytometer (BD, usa) showed over 90% purity. For samples/leukemia grafts that do not respond well (e.g., reduced in number) to compound D, LDA was performed using only cells from RF or BM given the higher leukemia population. Mouse Cell Depletion was performed according to the instructions provided by Miltenyi Biotec (Mouse Cell Depletion Kit, catalog No. 130-.

Treatment and assay procedures: mice were euthanized 10 to 12 weeks after the secondary transplantation, and both injected (RF) and non-injected Bone Marrow (BM) were collected to flush bone marrow cells for suspension. AML engraftment with and without injected bone marrow was analyzed by flow cytometry using human specific antibodies. Cells harvested from injected and non-injected bone marrow from each treatment group were pooled and live frozen for future analysis.

Cells harvested from the injected right femur and non-injected femur were stained with mouse anti-human antibody as described above. After staining, the washed cells were run on a LSRII flow cytometer (BD, usa). A total of 10,000 to 20,000 events were collected per sample. The collected data were analyzed by FlowJo software to assess AML engraftment levels in different tissues as determined by the percentage of human CD45+ CD33+ cells. LSC frequencies between vehicle and compound D treated groups were analyzed and compared using WEHI bioinformatics ELDA software (bioinf.

6.4.3.2. Data analysis

Implantation of AML in injected and non-injected femurs was analyzed by flow cytometry. Charts were generated using GraphPad Prism software and statistically analyzed. Statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Tukey multiple post-comparison tests.

6.4.4. Effect of Compound D on eradication of acute myeloid leukemia blasts in xenografts comprising leukemia Stem cells

All samples were grouped based on the 17-gene score described above. In both RF and non-BM injections, most samples were considered to be responders or partial responders to compound D (29 out of 35 samples), including samples with high LSC17 scores. Since AML samples with a high LSC17 score are typically low responders to standard chemotherapy with poor prognosis, this data suggests that compound D can target leukemia with poor prognosis and these patients may be good candidates for clinical trials to treat patients with refractory and relapsed leukemia.

To determine whether quiescent LSCs were resistant to compound D, a secondary transplantation of high responder AML cells was performed. High responders were identified as samples in which leukemic cells were barely detectable in the bone marrow of one mouse after compound D treatment. If rare resting LSCs are maintained in leukemic cells remaining after compound D administration, these resting LSCs may become active and re-lodge in secondary mice after undergoing serial transplantation. LSC17 high samples of four AML blasts eradicated by compound D treatment were selected for secondary transplantation by injecting pooled bone marrow cells collected from 5 primary mice treated with vehicle or compound D into 5 secondary mice (see table 9). In this way, cells collected from one primary mouse were injected into one secondary mouse without any dilution of the cell dose or loss of any residual cells. Cells harvested from primary mice transplanted with cells from

AML patients

130311 and 130826 did not proliferate/re-lodge in any secondary mice (from vehicle and compound D treated primary mice). Cells from AML patients 150238 harvested from vehicle-treated primary mice were re-populated into only one of 5 secondary mice. Cells from AML patient 150238 harvested from compound D treated primary mice did not re-lodge in any secondary mice, and cells from secondary patients harvested from vehicle treated primary mice re-lodged in only one of 5 mice. While cells from AML patient 110625 harvested from vehicle-treated primary mice repopulated all 5 transplanted secondary mice, cells harvested from compound D-treated primary mice repopulated the bone marrow of one secondary mouse. Furthermore, no leukemic cells from compound D-treated mice were found in the bone marrow of the transplanted secondary mice, indicating that in this sampling of compound D responders, LSCs with self-renewal capacity were targeted by compound D.

Table 9: secondary transplantation of acute myeloid leukemia cells from vehicle and Compound D-treated Primary xenograft mice

AML ═ acute myeloid leukemia; BM ═ bone marrow (not injected); ID is identification; hi is high; LDA ═ limiting dilution assay; LSC ═ leukemic stem cells; LSC17 ═ leukemia stem cell 17-gene score; no. number; RF ═ right femur (injection); veh is vehicle.

^a In one secondary mouse with LSC engraftment after injection of AML cells from compound D-treated one mouse, LSCs were detected only in non-injected bone marrow and not RF (injected AML cells).

Acute myeloid leukemia cells were isolated from one injection of vehicle or compound D treated mice (both from RF and BM) identified as responders and injected into secondary mice without LDA. Mice injected with more than 1% of total bone marrow cells in the right femur with CD45+ CD33+ human leukemia cells will be considered engrafted with AML LSC.

6.4.5. Limiting dilution assay to demonstrate depletion of leukemic stem cells by Compound D in reaction samples

Serial transplantation and limiting dilution assays were performed on samples with residual human AML cells to determine whether compound D reduced the frequency of LSCs in mice once. Human leukemia cells were first purified from the bone marrow depleted mouse bone marrow cells from the right femur and non-injected bone marrow of the harvested mice once. Purified human leukemia cells were transplanted intrafemtocarpally into secondary NOD/SCID mice at the same cell number for vehicle and compound D treatment groups. For each treatment group of each patient sample, at least 4 different cell doses were used for the secondary LDA-determination. Compound D was not administered to secondary mice. Secondary mice were sacrificed around 12 weeks post-transplantation to assess the level of AML engraftment at each cell dose. Mice injected with more than 1% of total bone marrow cells in the right femur with CD45+ CD33+ human leukemia cells were considered to be engrafted with AML LSC. LSC frequencies between vehicle and compound D treated groups were analyzed and compared using WEHI bioinformatics ELDA software (bioinf. Representative secondary migration LDAs are shown in table 10, table 11, and fig. 19 to detail how LDAs were analyzed with ELDA software. Cells of AML patient 130578 harvested from vehicle or compound D treated primary mice were transplanted into secondary mice at the indicated cell dose (table 10). The number of transplanted and implanted mice at each cell dose for each treatment group is summarized (see table 10) and entered into the ELDA software to calculate LSC frequency.

Table 10: representative secondary graft limiting dilution assay: injection and implantation results

No. number.

Serial dilutions of acute myeloid leukemia cells from acute myeloid leukemia patient 130578 isolated from primary vehicle or compound D treated mice are shown transplanted and implanted into secondary mice.

Confidence intervals for group mean LSC frequencies are shown in table 11, where LSC frequencies were estimated as 1 LSC out of 14,536 cells in vehicle-treated one mouse and 1 LSC out of 136,136 cells in compound D-treated one mouse; LSC frequency was significantly reduced by 9.4 fold (p ═ 6.2x 10) after compound D treatment compared to vehicle ^-5 ). In the representative confidence interval plot shown in fig. 19, the red line represents the estimated group mean LSC frequency in the vehicle control, and the solid line represents the estimated group mean LSC frequency in the compound D-treated one-time mice. These data indicate that compound D significantly eliminated LSCs with self-renewal capacity in AML patients 130578 compared to vehicle controls.

Table 11: representative secondary graft limiting dilution assay: confidence intervals for leukemic stem cell frequency isolated from primary mice

Group of	Lower confidence level	Estimating frequency	Upper limit confidence level
				Compound D	361399	136136***	51281
Media	30952	14536	6827

P <0.001, relative to vehicle control group.

The estimated frequency of Leukemic Stem Cells (LSCs) from compound D or vehicle-treated mice acute myeloid leukemia patient 130578 was calculated using Walter and Eliza Hall Institute limiting dilution analysis software (available from bio if. wenhi. edu. au) and shown as 1 LSC per total cell (e.g., estimated as 1 LSC per 136,136 cells for compound D).

The results of secondary transplantation of LDA from 16 AML patient samples (including 4 samples [110500, 120846, 100348, and 09191] of LDA in the first pilot study; section 6.1) are summarized in Table 12. Three samples failed to re-lodge in secondary mice, including one responder (120791) and two non-responders (90191 and 140171). The other 13 samples were able to re-enter the secondary mice, including 8 samples that were responsive to compound D in the primary mice, 2 samples that were partially responsive to compound D, and 3 non-responders. Six samples had reduced LSC frequency after compound D administration, with a reduction level in one mouse from 2.1-fold to 13.3-fold. LDA of AML responder patient 130926 showed that compound D did not reduce LSC frequency, probably because the starting cell dose was too low. The highest cell dose of LDA for this patient was only 200,000 cells/mouse, since compound D eliminated most of the AML cells in one xenograft mouse, and only one mouse in each treatment group was re-populated at this cell dose. The frequency of LSCs increased (1.6-fold and 12.6-fold, respectively) in one mouse after compound D treatment in samples obtained from

AML patients

110484 and 130695 compared to other responders. For 3 non-responders, AML patient 120858 was an aggressive sample and all mice were re-populated with 2000 cells/mouse (lowest LDA dose), so a dose of less than 2000 cells would need to be transplanted to determine the LSC frequency in vehicle and compound D treated mice. Compound D did not reduce the frequency of LSC in the other 2 non-responsive samples (samples of

AML patients

120860 and 120846 increased 1.3-fold and 1.8-fold, respectively).

BM ═ bone marrow (not injected); ID is identification; LSC ═ leukemic stem cells; LSC17 ═ leukemia stem cell 17-gene score; NA is not applicable; NC — not calculated; ND is not determined; NR is a non-responder; PR ═ partial responders; r is the reactant; RF ═ right femur (injection); veh is vehicle. P < 0.001.

^a All mice transplanted with the two treatment groups with the lowest cell dose were implanted, so fold change in LSC frequency was not applicable.

^b The minus sign indicates an increase in fold change.

Acute Myeloid Leukemia (AML) cells were isolated from one mouse treated with Acute Myeloid Leukemia (AML) cells injection with vehicle or compound D and evaluated for LSC17 score (LSC17 hi ≧ 0.50) and in vivo responsiveness to compound D based on reduction of AML cells compared to vehicle control. Patient samples were classified based on the percent reduction of AML cells relative to vehicle in RF or BM after compound D treatment (R ═ 60%; PR ═ 30% -60%; NR ═ 30%). Cells (from RF, BM, or both) were diluted in LDA and injected into irradiated secondary mice, where the LSC frequency was determined using Walter and Eliza Hall Institute extreme limited dilution analysis software (bio if. wehi. edu. au) and shown as 1 LSC per total cell. Fold-frequency changes from compound D were determined relative to vehicle control and evaluated by one-way analysis of variance. P-values represent the comparison of LSC frequency change for compound D treatment versus control.

6.4.6. Conclusion

After compound D treatment of NOD/SCID mice bearing human AML, secondary transplants were performed with LDA to determine if compound D could target LSC. Most reactive leukemia samples had LSCs targeted by compound D and the reduction in frequency was different (6 out of 10 samples). The frequency of LSCs was not decreased in one responder and increased in 2 responders to compound D treatment. Compound D did not reduce LSC frequency in 3 non-responders who re-inhabit the secondary mice. Overall, these results indicate that compound D not only targets leukemic blasts of responders in a human AML mouse model, but also reduces LSCs with self-renewal capacity. The observation that compound D does not target LSCs in all AML samples reflects the resistance and heterogeneity of response of LSCs in AML to compound D.

From the foregoing, it will be appreciated that, although specific embodiments have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope provided herein. All references mentioned above are incorporated herein by reference in their entirety.

Claims

1. A method of identifying a subject with Acute Myeloid Leukemia (AML) that is likely to respond to a treatment comprising a compound, or predicting the responsiveness of a subject with or suspected of having AML to a treatment comprising the compound, the method comprising:

i. Providing a sample from the subject;

measuring the gene expression level of one or more genes in the sample;

identifying the subject as likely to be responsive to a treatment comprising the compound if the level of the LSC feature score is above its reference level,

wherein the compound is 2- (4-chlorophenyl) -N- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2, 2-difluoroacetamide having the structure (Compound D):

2. A method of treating a subject having AML with a compound, the method comprising:

i. providing a sample from the subject;

measuring the gene expression level of one or more genes in the sample;

(b) Administering a therapeutically effective amount of the compound to the subject if the subject is identified as likely to respond to a treatment comprising the compound,

wherein the compound is compound D or a stereoisomer or a mixture of stereoisomers thereof, an isotopologue, a pharmaceutically acceptable salt, a tautomer, a solvate, a hydrate, a co-crystal, a clathrate, or a polymorph.

3. The method of claim 1 or claim 2, wherein the LSC signature score is calculated as a weighted sum of the expression levels of the one or more genes.

4. The method of any one of claims 1 to 3, wherein the reference level is the median LSC feature score in a population.

5. The method of any one of claims 1 to 3, wherein the reference level is a predetermined LSC feature score level.

6. The method of any one of claims 1 to 5, wherein a LSC signature score above its reference level indicates that the subject has resistant and/or refractory AML.

7. The method of any one of claims 1 to 6, wherein the one or more genes are selected from

(a) CD34, SPINK2, LAPTM48, HOXA5, GUCY1A3, SHANK3, ANGPT1, ARHGAP22, LOC284422, MYCN, MAMDC2, PRSSL1, KIAA0125, GPSM1, HOXA9, MMRN1, FSCN1, DNMT38, HOXA6, AIF1L, SOCS2, CDK6, FAM69B, NGFRAP1, C3orf54, CPXM1, TNFRSF4, ZBTB46, DPYSL3, NYLNRIN, COL 1, FAM30A, C10orf 647f 140, SPNS A, GPR 4, AKR1C A, FLT 4, TFPI A, KC A, ADPR A, 685A, 685-S, A

(b) CD34, SPINK2, LAPTM48, HOXA5, GUCY1A3, SHANK3, ANGPT1, ARHGAP22, LOC284422, MYCN, MAMDC2, PRSSL1, KIAA0125, GPSM1, HOXA9, MMRN1, FSCN1, DNMT38, HOXA6, AIF1L, SOCS2, CDK6, FAM69B, NGFRAP1, C3orf54, CPXM1, TNFRSF4, ZBTB4, DPYSL 4, NYRRIN, COL24A 4, FAM30 4, C10orf140, SPNS 4, GPR 4, AKR1C 4, FLT 4, TFPI 4, KCR 4, C150, VWF 4, ATP 4, ATP 4, 4 and AFVM 4.

8. The method of any one of claims 1 to 6, wherein the one or more genes are selected from AKR1C3, ARHGAP22, CD34, CDK6, CPXM1, DNMT3B, DPYSL3, EMP1, GPR56, KIAA0125, LAPTM4B, MMRN1, NGFRAP1, NYRRIN, SMIM24, SOCS2, and ZBTB 46.

9. The method of any one of claims 1 to 6, wherein the LSC trait score is based on gene expression levels of AKR1C3, ARHGAP22, CD34, CDK6, CPXM1, DNMT3B, DPYSL3, EMP1, GPR56, KIAA0125, LAPTM4B, MMRN1, NGFRAP1, NYRRIN, SMIM24, SOCS2, and ZBTB 46.

10. The method of any of claims 1 to 6, wherein the LSC feature score is calculated as follows: (the expression level of DNMT3B × the weight of DNMTT 3B) + (the expression level of ZBTB46 × the weight of ZBTB 46) + (the expression level of NYNRIN × the weight of NYNRIN) + (the expression level of ARHGAP22 × the weight of ARHGAP 22) + (the expression level of lamm 4B × the weight of lamm 4B) + (the expression level of MMRN1 × the weight of MMRN 1) + (the expression level of DPYSL 1 × the weight of DPYSL 1) + (the expression level of KIAA0125 × the weight of KIAA 0125) + (the expression level of CDK 1 × the weight of CDK 1) + (the expression level of CPXM1 × the weight of CPXM 1) + (the expression level of SOCS 1 × the weight of SOCS 1) + (the expression level of SMIM 1 × the weight of SMIM 1) + (the weight of emgpr 1 × the expression level of emgpr 1 × the weight of SOCS 1) + (the expression level of samakc 1 × the expression level of samm 1) + (the weight of samr 1) + (the expression level of samgpr 1 × the weight of samm 1) + (the weight of ZBTB 1) + (the expression level of ZBTB 1 × the weight of ZBTB 1) + (the expression level of ZBTB 1) + (the weight of ZBTB 1) + (the expression level of ZBTB 1) + (the weight of ZBTB 1); and is

Wherein DNMTT3B is within the range of 0.08 to 0.09, ZBTB46 is within the range of-0.03 to-0.04, NYRNIN is within the range of-0.008 to 0.009, ARHGAP22 is within the range of-0.015 to 0.01, LAPTM4B is within the range of-0.006 to 0.005, MMRN1 is within the range of 0.02 to 0.03, DPYSL3 is within the range of 0.02 to 0.03, KIAA0125 is within the range of 0.01 to 0.02, CDK6 is within the range of-0.08 to-0.07, CPXM1 is within the range of-0.02 to-0.03, SOCS2 is within the range of 0.02 to 0.03, SMM 3 is within the range of 0.02 to 0.03, SMIM 3 is within the range of 0.04 to 0.05, EMBR 3 is within the range of-0.04 to 0.05, and CRP is within the ranges of-0.0550.04, and GPR, and KR 4 is within the ranges of-0.05 to 0.055, and is within the ranges of 0.02 to 0.03.

11. The method of any of claims 1 to 6, wherein the LSC feature score is calculated as follows: (expression level of DNMT3B × 0.0874) + (expression level of ZBTB46 × -0.0347) + (NYNRIN × 0.00865) + (ARHGAP22 × -0.0138) + (lamm 4B expression level × 0.00582) + (MMRN1 × 0.0258) + (DPYSL3 expression level × 0.0284) + (KIAA0125 expression level × 0.0196) + (CDK6 expression level of × -0.0704) + (CPXM1 expression level × -0.0258) + (SOCS2 × 0.0271) + (SMIM24 expression level × -0.0226) + (EMP1 expression level × 0.0146) + (NGFRAP1 × 0.0465) + (CD34 expression level × 0.0338) + (AKR1 × 3) + (akgpr × 3646) + (akgpr 3646).

12. The method of claim 11, wherein the reference level is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8.0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.

13. The method of any one of claims 1 to 6, wherein the LSC signature score is based on gene expression levels of TNFRSF4, SLC4A1, SLC7A7, and AIM 2.

14. The method of claim 13, wherein the LSC feature score is calculated as follows: (expression level of TNFRSF4 × weight of TNFRSF 4) + (expression level of SLC4a1 × weight of SLC4a 1) + (expression level of SLC7a7 × weight of SLC7a 7) + (expression level of AIM2 × weight of AIM 2); and wherein TNFRSF4 is weighted in the range of-1.5 to-1, SLC4A1 is weighted in the range of 13 to 14, SLC7A7 is weighted in the range of-4 to-3, and AIM2 is weighted in the range of-3 to-4.

15. The method of claim 13, wherein the LSC feature score is calculated as follows: (expression level of TNFRSF4 × -1.13) + (expression level of SLC4A1 × 13.59) + (expression level of SLC7A7 × -3.57) + (expression level of AIM2 × -3.04).

16. The method of claim 15, wherein the reference level is in the range of-50 to 115, -45 to 110, -40 to 105, -37 to 100, -30 to 95, -25 to 90, -20 to 85, -15 to 80, -10 to 75, -5 to 70, 0 to 65, 5 to 60, 10 to 55, 15 to 50, 20 to 45, 25 to 40, or 30 to 35.

17. The method of any of claims 1 to 6, wherein the LSC signature score is based on gene expression levels of SLC4A1, SLC7A7, and AIM 2.

18. The method of claim 17, wherein the LSC feature score is calculated as follows: (SLC4a1 expression level x weight of SLC4a 1) + (SLC7a7 expression level x weight of SLC7a 7) + (AIM2 expression level x AIM2 weight); and wherein SLC4A1 is weighted in the range of 11 to 15, SLC7A7 is weighted in the range of-5.5 to-1.5, and AIM2 is weighted in the range of-5 to-1.

19. The method of claim 17, wherein the LSC feature score is calculated as follows:

the calculation is as follows: (SLC4A1 expression level X13.59) + (SLC7A7 expression level X-3.57) + (AIM2 expression level X-3.04).

20. The method of claim 19, wherein the reference level is in the range of-65 to 110, -60 to 105, -55 to 100, -49 to 93, -45 to 90, -40 to 85, -35 to 80, -30 to 75, -25 to 70, -20 to 65, -15 to 60, -10 to 55, -5 to 50, 0 to 45, 5 to 40, 10 to 35, 15 to 30, 20 to 35, or 25 to 30.

21. A method of identifying a subject having AML who is likely to respond to a treatment comprising a compound or predicting the responsiveness of a subject having or suspected of having AML to a treatment comprising the compound, the method comprising:

i. Providing a sample from the subject;

administering the compound to the sample;

measuring the proportion of one or more cell types;

identifying the subject as likely to be responsive to a treatment comprising the compound if the proportion of the one or more cell types is different from the reference proportion of the cells,

22. A method of treating a subject having AML with a compound, the method comprising:

i. providing a sample from the subject;

administering the compound to the sample;

measuring the proportion of one or more cell types;

23. The method of claim 21 or claim 22, wherein the reference proportion of one cell type is the proportion of the cell type in the sample prior to administration of the compound.

24. The method of claim 21 or claim 22, wherein the reference proportion of one cell type is a predetermined proportion.

25. The method according to any one of claims 21 to 24, wherein the method comprises measuring the proportion of primitive cells and/or the proportion of differentiated leukemic cells.

26. The method of claim 25, wherein a decrease in the proportion of blasts and/or an increase in the proportion of differentiated leukemia cells as compared to their respective proportions prior to administration of the compound indicates that the subject is likely to respond to a treatment comprising the compound.

27. The method of any one of claims 21 to 26, wherein the method comprises measuring the proportion of CD34+, CD15+, CD14+ and/or CD11b + cells.

28. The method of any one of claims 21 to 27, wherein the method comprises measuring the proportion of CD34+ cells, and wherein a decrease in the proportion of CD34+ cells as compared to the proportion of CD34+ cells prior to administration of the compound indicates that the subject is likely to be responsive to a treatment comprising the compound.

29. The method of any one of claims 21 to 27, wherein the method comprises measuring the proportion of CD15+ cells and/or CD14+ cells, and wherein an increase in the proportion of CD15+ cells and/or CD14+ cells as compared to the proportion of CD15+ cells and/or CD14+ cells prior to administration of the compound indicates that the subject is likely to be responsive to a treatment comprising the compound.

30. The method according to any one of claims 1 to 29, wherein the AML is refractory or resistant.

31. The method according to any one of claims 1 to 29, wherein the AML is resistant to treatment with one or more agents selected from daunomycin, cytarabine (ara-C) and gemtuzumab ozogamicin or resistant to chemotherapy.